Fusion proteins for delivery of gdnf to the cns

ABSTRACT

The invention provides compositions, methods, and kits for increasing transport of GDNF across the blood brain barrier while allowing its activity to remain substantially intact. The GDNF is transported across the blood brain barrier via one or more endogenous receptor-mediated transport systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/828,557, filed Mar. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 14/144,460, filed Dec. 30, 2013, now abandoned,which is a continuation of U.S. patent application Ser. No. 12/323,232filed Nov. 25, 2008, now U.S. Pat. No. 8,741,260, issued Jun. 3, 2014,which is a continuation-in-part of U.S. patent application Ser. No.11/245,546 filed Oct. 7, 2005, now U.S. Pat. No. 8,142,781, issued Mar.27, 2012, and which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/990,290 filed Nov. 26, 2007, the contents ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML, format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jul. 11, 2022, isnamed 28570-706_303_SL.xml and is 97,000 bytes in size.

BACKGROUND OF THE INVENTION

Neurological disorders represent a major cause of mortality anddisability worldwide. Despite extensive progress, current treatmentoptions remain limited in some aspects. One major reason for thislimitation is that the brain is unique in allowing only select access tomolecules. While this is a useful protective mechanism, it also meansthat many potentially beneficial molecular entities do not have accessto the central nervous system (CNS), and thus are unable to exert atherapeutic effect in many neurological disorders or other conditions ofthe CNS. The present invention represents an advance in providingaccessibility of the CNS for molecular entities whose ability to crossthe blood brain barrier is limited.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition comprising aneurotherapeutic peptide comprising an amino acid sequence which is atleast 80% (e.g., 95%) identical to the amino acid sequence of maturehuman GDNF (SEQ ID NO:52) covalently linked to a structure that iscapable of crossing the blood brain barrier (BBB) (e.g., an antibody).In some embodiments, the structure that is capable of crossing the BBBcrosses the BBB on an endogenous BBB receptor. In some embodiments, theendogenous BBB receptor is the insulin receptor, transferrin receptor,leptin receptor, lipoprotein receptor, and the IGF receptor. In someembodiments, the structure that is capable of crossing the BBB is amonoclonal antibody. In some embodiments, the monoclonal antibody is achimeric monoclonal antibody. In one embodiment, the chimeric antibodycontains sufficient human sequences to avoid significant immunogenicreaction when administered to a human In some embodiments, theabove-mentioned neurotherapeutic peptide is covalently linked at itsamino terminus to the carboxy terminus of the heavy chain of the MAb. Inone embodiment, the neurotherapeutic peptide of the above-mentionedcomposition comprises the amino acid sequence of mature human GDNF.

In a further aspect provided herein is a composition for treating aneurological disorder comprising a neurotherapeutic peptide comprisingan amino acid sequence which is at least 80% identical to the amino acidsequence of mature human GDNF covalently linked to an immunoglobulinthat is capable of crossing the blood brain barrier, wherein thecomposition is capable of crossing the BBB in an amount that iseffective in treating the neurological disorder. In some embodimentsprovided herein is a mammalian cell comprising the just-mentionedcomposition.

In another aspect provided herein is a method of transport of aneurotherapeutic peptide comprising an amino acid sequence which is atleast 80% identical to the amino acid sequence of mature human GDNF (SEQID NO:52) from the peripheral circulation across the BBB in an effectiveamount, comprising peripherally administering to an individual the GDNFcovalently attached to a structure that crosses the BBB, underconditions where the agent covalently attached to a structure thatcrosses the BBB is transported across the BBB in an effective amount.

In yet another aspect provided herein is a method for treating a CNSdisorder in an individual comprising peripherally administering to theindividual an effective amount of a composition comprising aneurotherapeutic peptide comprising an amino acid sequence which is atleast 80% (e.g., 95%) identical to the amino acid sequence of human GDNFcovalently attached to a structure capable of crossing the BBB. In oneembodiment, the neurotherapeutic peptide comprises the amino acidsequence of human GDNF. In some embodiments, the administering is oral,intravenous, intramuscular, subcutaneous, intraperitoneal, rectal,transbuccal, intranasal, transdermal, or inhalation administration. Insome embodiments, the CNS disorder is an acute CNS disorder (e.g.,spinal cord injury, brain injury focal brain ischemia and global brainischemia). In other embodiments, the CNS disorder is a chronic CNSdisorder (e.g., a chronic neurodegenerative disease, drug addiction, oralcohol addiction). In some embodiments, the chronic neurodegenerativedisease is Parkinson's disease or a motor neuron disease (e.g.,amyotrophic lateral sclerosis). In some embodiments, the individual tobe treated is administered about 1 to about 100 mg of the compositionused in above-mentioned method.

In yet another aspect provided herein is a composition comprising aneurotherapeutic peptide comprising an amino acid sequence which is atleast 80% identical to the amino acid sequence of human GDNF covalentlylinked to an immunoglobulin, wherein the neurotherapeutic peptide has aserum half-life that is an average of at least about 5-fold greater thanthe serum half-life of the neurotherapeutic peptide alone. In someembodiments, the immunoglobulin is an antibody to an endogenous BBBreceptor (e.g., the insulin receptor, transferrin receptor, leptinreceptor, lipoprotein receptor, or the IGF receptor). In one embodiment,the neurotherapeutic peptide comprises the amino acid sequence of humanGDNF.

In a further aspect provided herein is a method for treating substanceabuse in an individual, comprising administering to the individual aneffective amount of a composition comprising a neurotherapeutic peptidecomprising an amino acid sequence which is at least 80% identical to theamino acid sequence of mature human GDNF (SEQ ID NO:52) covalentlyattached to a structure capable of crossing the BBB.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 . Diagram showing genetic engineering of a eukaryotic expressionvector encoding a fusion gene comprised of the variable region of theheavy chain (VH) of the chimeric HIRMAb, a genomic fragment encoding theconstant region of human IgG1, which is comprised of 4 regions (CH1,hinge, CH2, and CH3), and the cDNA for the BDNF variant (vBDNF).Transcription of the gene is driven by the human IgG1 promoter (PRO).This vector produces the heavy chain (HC) of the fusion protein.

FIG. 2 . Diagram showing genetic engineering of a bacterial expressionplasmid encoding vBDNF cDNA with modified 5′- linker (FIG. 2A) andmodified 3′-linker (FIG. 2B).

FIG. 3 . Ethidium bromide stained agarose gels showing size of variousconstructs that are intermediates in construction of a tandem vectorthat produces the fusion protein. (A) Lanes 1-2: plasmid from FIG. 2digested with NruI showing 0.4 kb vBDNF and 3.5 kb vector backbone. Lane3: MW size standards ranging from 1.4-0.1 kb. Lane 4: MW size standardsranging from 23-0.6 kb. (B) Lane 1: the 0.4 kb vBDNF cDNA is produced bythe polymerase chain reaction (PCR) using cDNA reverse transcribed frompolyA+ RNA isolated from human U87 glioma cells; the PCR primersequences are given in Table 2. Lanes 2 and 3: same MW size standards asshown in panel A. (C) lane 1: clone 416 following digestion with NheIand BamHI; lane 2: negative clone; lane 3: clone 400 following digestionwith NheI and BamHI: lanes 4 and 5: same MW size standards as shown inpanel A. (D) PCR fragments of DNA encoding fusion protein HC (lane 1)and LC (lane 2); lanes 3-4: same MW size standards as shown in panel A.(E) lanes 1-4: 4 different but identical copies of clone 422a followingdigestion with NheI, showing release of 0.4 kb fusion protein HCvariable region (VH) cDNA; lanes 5-6: same MW size standards as shown inpanel A. (F) lanes 1-4: 5 different but identical copies of clone 423afollowing digestion with EcoRV and BamHI, showing release of 0.7 kbentire LC cDNA; lanes 5-6: same MW size standards as shown in panel A.(G) Restriction endonuclease mapping of tandem vector (FIG. 12 ) withPvuI (lane 1), and EcoRI-HindIII (lane 2). PvuI (single cut) producedthe expected linear DNA band of ˜11 kb. Digestion with EcoRI and HindIIIreleases both the fusion protein light chain (i.e. 1.8 kb) and DHFR(i.e. 1.5 kb) expression cassettes. The ˜8 kb band represents thebackbone vector with the fusion protein heavy chain expression cassette;lanes 3-4: same MW size standards as shown in panel A, albeit in reverseorder.

FIG. 4 . Nucleotide (SEQ ID NO: 21) and amino acid (SEQ ID NO: 22)sequence of fusion site between carboxyl terminus of the fusion proteinHC and the amino terminus of the vBDNF. The 3-amino acid linker betweenthe HIRMAb HC and the vBDNF is shown, as well as the new stop codon atthe carboxyl terminus of vBDNF.

FIGS. 5A and 5B. Nucleotide sequence (SEQ ID NO: 23) of fusion proteinHC gene cloned into plasmid 416. Italics: human IgG1 constant regionintrons; bold font: human IgG1 exon sequence; underline font: vBDNF.

FIG. 6 . Amino acid sequence (SEQ ID NO: 24) of the fusion protein HC.The 19 amino acid signal peptide is underlined, as is the 3-amino acidlinker between the CH3 region and the vBDNF. The N-linked glycosylationconsensus sequence within CH2 is underlined.

FIG. 7 . The amino acid sequence (SEQ ID NO: 25) of the differentdomains of the fusion protein HC are shown. FIG. 7 discloses the “SignalPeptide” sequence as residues 1-19 of SEQ ID NO: 25, the “FR1” sequenceas residues 20-44 of SEQ ID NO: 25, the “CDR1” sequence as residues45-54 of SEQ ID NO: 25, the “FR2” sequence as residues 55-68 of SEQ IDNO: 25, the “CDR2” sequence as residues 69-85 of SEQ ID NO: 25, the“FR3” sequence as residues 86-117 of SEQ ID NO: 25, the “CDR3” sequenceas residues 118-121 of SEQ ID NO: 25, the “FR4” sequence as residues122-132 of SEQ ID NO: 25, the “CH1” sequence as residues 133-230 of SEQID NO: 25, the “Hinge” sequence as residues 231-242 of SEQ ID NO: 25,the “CH2” sequence as residues 243-355 of SEQ ID NO: 25, the “CH3”sequence as residues 356-462 of SEQ ID NO: 25, the “linker” sequence asresidues 463-465 of SEQ ID NO: 25 and the “vBDNF” sequence as residues466-582 of SEQ ID NO: 25.

FIG. 8 . Diagram showing production of the intronless eukaryoticexpression vector, clone 422a, which encodes the fusion protein HC. Thefusion protein HC cDNA was produced by PCR from cDNA generated byreverse transcriptase of RNA isolated from myeloma cells transfectedwith clone 416. FIG. 8 , Part A, indicates the step of obtaining mRNAfrom cells transfected with the fusion protein HC gene; FIG. 8 , part B,indicates the step of obtaining the cDNA for the fusion protein HC geneby RT-PCR.

FIGS. 9A-9C. (FIG. 9A) Nucleotide sequence (SEQ ID NO: 26) of the fusionprotein HC cDNA inserted in clone 422a. (FIGS. 9B and 9C) Amino acidsequence (SEQ ID NO: 28) of the fusion protein HC that is deduced fromthe nucleotide sequence (SEQ ID NO: 27) shown in panel A. The sequenceof the signal peptide is underlined.

FIG. 10 . Diagram showing production of the intronless eukaryoticexpression vector, clone 423a, which encodes the fusion protein LC. Thefusion protein LC cDNA was produced by PCR from cDNA generated byreverse transcriptase of RNA isolated from myeloma cells transfectedwith an expression vector producing the LC gene that was derived fromchromosomal fragment encoding intron/exon sequence of the human kappa LCgene with the VL of the chimeric HIRMAb LC. FIG. 10 , part A, indicatesthe step of obtaining mRNA from cells transfected with the fusionprotein LC gene; FIG. 10 , part B, indicates the step of obtaining thecDNA for the fusion protein LC gene by RT-PCR.

FIGS. 11A and 11B. (FIG. 11A) Nucleotide sequence (SEQ ID NO: 29) of thefusion protein LC cDNA inserted in clone 423a. (FIG. 11B) (SEQ ID NOS 30& 31) Amino acid sequence of the fusion protein LC that is deduced fromthe nucleotide sequence shown in panel A. The sequence of the signalpeptide is underlined.

FIG. 12 . Diagram showing the construction of a tandem vector encodingthe HC and LC genes of the fusion protein. The TV was engineered fromthe cDNA expression vectors, clones 422a and 423a, for the HC and LC,respectively, as well as from a bacterial expression plasmid encodingthe expression cassette for mouse DHFR.

FIGS. 13A-13D. Nucleotide sequence (SEQ ID NO: 32) of the fusion proteinHC gene expression cassette (Nucleotides 1-2,856 of SEQ ID NO: 32) andLC gene expression cassette (Nucleotides 2,857-4,720 of SEQ ID NO: 32),and the DHFR gene expression cassette (Nucleotides 4,732-6,505 of SEQ IDNO: 32) incorporated in the tandem vector.

FIGS. 14A and 14B. Deduced amino acid sequence (SEQ ID NO: 34) of thefusion protein HC based on tandem vector nucleotide sequence (SEQ ID NO:33) analysis. The signal peptide sequence is underlined.

FIG. 15 . Deduced amino acid sequence of the fusion protein LC based ontandem vector nucleotide sequence analysis (SEQ ID NO 35 & 36). Thesignal peptide sequence is underlined.

FIG. 16 . Deduced amino acid sequence of the DHFR based on tandem vectornucleotide sequence analysis (SEQ ID NO 37 & 38).

FIG. 17 . Viable and total cell density of CHO cells in bioreactormaintained continuously for 50 days; the CHO cells had been stablytransfected with the tandem vector encoding the fusion protein.

FIG. 18 . Structure of fusion protein, a bi-functional molecule thatboth (a) binds to the human BBB human insulin receptor (HIR) to enabletransport across the BBB from blood, and (b) binds to the trkB onneurons to induce neuroprotection.

FIG. 19 . Correlation of 2 different ‘sandwich’ immunoassays, where thesecondary antibody is either directed against the Fc region of humanIgG1 (x-axis) or against human BDNF (y-axis). The primary antibody ineither assay is directed against the human kappa light chain Themeasured level of fusion protein in CHO cell conditioned medium is thesame whether the anti-Fc or the anti-BDNF antibody is used.

FIG. 20 . Reducing (left) and non-reducing (right) sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of chimeric HIRMAband fusion protein. Under reducing conditions, the size of the lightchain, 30 kDa, is identical for chimeric HIRMAb and the fusion protein;the size of the heavy chain of fusion protein is about 15 kDa largerthan the chimeric HIRMAb heavy chain, owing to the presence of the BDNF.Under non-reducing conditions, the chimeric HIRMAb and the fusionprotein migrate as single hetero-tetrameric species with molecularweights of 180 and 200 kDa, respectively.

FIG. 21 . (Left panel) Western blot with anti-human IgG primaryantibody. The size of the heavy chain of the fusion protein and thechimeric HIRMAb is 64 kDa and 50 kDa, respectively, and the size of thelight chain for either the fusion protein or the chimeric HIRMAb is 25kDa. (Right panel) Western blot with anti-human BDNF antibody, whichreacts with either fusion protein or BDNF, but not with chimeric HIRMAb.MW standards (STDS) are shown on the right side.

FIG. 22 . Isoelectric focusing (IEF) of isoelectric point (pI) standards(lane 1), chimeric HIRMAb (lanes 2 and 4), BDNF (lane 3), and fusionprotein (lane 5). Whereas BDNF is highly cationic with a pI>10, the pIof the fusion protein approximates the pI of the chimeric HIRMAb, whichis about 8.5, and close to the theoretical pI of the fusion protein.

FIG. 23 . (A) Outline for human insulin receptor (HIR) competitiveligand binding assay (CLBA). The HIR extracellular domain (ECD) is boundby the [¹²⁵I]-labeled murine HIRMAb, and this binding is competitivelydisplaced by either the chimeric HIRMAb or the fusion protein, as shownin Panel B. (B) Displacement of binding of [¹²⁵I]-labeled murine HIRMAbto the HIR ECD by either chimeric HIRMAb or fusion protein. The affinityof the chimeric HIRMAb to the HIR ECD is high, and the affinity of thefusion protein for the HIR ECD is not significantly different from thatof the chimeric HIRMAb. These results show that the fusion of the vBDNFto the carboxyl terminus of the chimeric HIRMAb heavy chain does notimpair binding of the fusion protein to the HIR.

FIG. 24 . (A) Design of trkB competitive ligand binding assay (CLBA).The advantage of the PEG linker is that this modification eliminates thehigh non-specific binding (NSB) of the cationic BDNF to the ELISA wells,which gives an assay with a high signal/noise ratio. The binding of theBDNF-PEG²⁰⁰⁰-biotin to the trkB ECD was detected with a peroxidasesystem using avidin and biotinylated peroxidase. (B) The binding of theBDNF-PEG²⁰⁰⁰-biotin to the trkB ECD is competitively displaced byrecombinant BDNF. This binding data was analyzed by non-linearregression analysis to yield the K₁ of BDNF binding, 3.5±1.3 pmol/welland the NSB parameter. (C) The binding of the BDNF-PEG²⁰⁰⁰-biotin to thetrkB ECD is competitively displaced by the fusion protein. This bindingdata was analyzed by non-linear regression analysis to yield the K₁ offusion protein binding, 2.2±1.2 pmol/well, which is not significantlydifferent than the K1 for native BDNF. These data show that the affinityof the fusion protein for the trkB receptor is equal to that of nativeBDNF.

FIG. 25 . (A) Design of hypoxia-reoxygenation neuroprotection assay inhuman neural SH-SY5Y cells. Exposure of the cells to retinoic acid for 7days causes an up-regulation in the gene expression of trkB, the BDNFreceptor. (B) Neuroprotection assay based on the measurement ofmitochondrial respiration with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).The maximal neuroprotection is established with 4 nM BDNF, and 4 nMfusion protein yields a comparable level of neuroprotection in humanneural cells. The MTT level does not return to that of non-hypoxiccells, because only about 50% of the cells induce trkB in response toretinoic acid.

FIG. 26 . (A) Light micrograph of capillaries isolated from human brain,used as an in vitro model system of the human BBB. (B) Radio-receptorassay of binding of [³H]-fusion protein to the HIR on the human BBB; thebinding is self-inhibited by unlabeled fusion protein. Fitting thesaturation data to a Scatchard plot with a non-linear regressionanalysis yields the binding parameters: K_(D)=0.55±0.07 nM,B_(max)=1.35±0.10 pmol/mg_(p).

FIG. 27 . Pharmacokinetics and brain uptake of fusion protein in theadult Rhesus monkey. (A) The serum concentration of [³H]-fusion protein,or [¹²⁵I]-murine HIRMAb, is plotted vs. time after a single intravenousinjection of either protein in anesthetized adult Rhesus monkeys. (B)The serum radioactivity that is precipitable by trichloroacetic acid(TCA) is plotted vs time after a single intravenous injection of either[³H]-fusion protein in the anesthetized adult Rhesus monkey, or[³H]-BDNF in the anesthetized adult rat. (C) Capillary depletionanalysis of brain distribution at 180 minutes after a single intravenousinjection of either [³H]-fusion protein, or [³H]-mouse IgG2a, in theanesthetized adult Rhesus monkey. (D) Primate brain concentrations offusion protein at 180 minutes after an intravenous injection of 373 μgfusion protein, as compared to the endogenous primate brainconcentration of BDNF.

FIG. 28 . Schematic illustration of the protein formed by fusion of theamino terminus of the mature GDNF to the carboxyl terminus of the CH3region of the heavy chain of the chimeric HIRMAb. The fusion protein isa bi-functional molecule: the fusion protein binds the HIR, at the BBB,to mediate transport into the brain, and binds the GFRα1, to mediateGDNF biologic effects in brain behind the BBB.

FIG. 29 . (A) Ethidium bromide stain of agarose gel of human GDNF cDNA(lane 1), which was produced by PCR from cDNA produced by reversetranscription of RNA from human U87 glial cells, and GDNF-specific ODNprimers (Table 6). Lane 2: DNA sizing standards (B) Genetic engineeringof pHIRMAb-GDNF, the eukaryotic expression plasmid encoding the fusionprotein of GDNF and the heavy chain (HC) of the chimeric HIRMAb. Thefusion gene is 5′-flanked by the cytomegalovirus (CMV) promoter and3′-flanked by the bovine growth hormone polyA (pA) sequence.

FIG. 30 . Reducing SDS-PAGE and Coomasie blue staining of protein Aaffinity purified, COS-cell chimeric HIRMAb and the HIRMAb-GDNF fusionprotein. Both are purified to homogeneity and are comprised of a heavychain and a light chain The molecular weight (MW) of the heavy chain(HC) of the HIRMAb-GDNF fusion protein is about 15 kDa larger than theMW of the HC of the chimeric HIRMAb, owing to fusion of the 15 kDa GDNF.

FIG. 31 . Western blot with either anti-human (h) IgG primary antibody(left) or an anti-human GDNF primary antiserum (right). Theimmunoreactivity of the COS cell derived HIRMAb-GDNF fusion protein iscompared to the chimeric HIRMAb and to recombinant GDNF. Both theHIRMAb-GDNF fusion protein and the HIRMAb have identical light chains onthe anti-hIgG Western. The HIRMAb-GDNF fusion heavy chain reacts withboth the anti-hIgG and the anti-human GDNF antibody, whereas the HIRMAbheavy chain only reacts with the anti-hIgG antibody. The size of theHIRMAb-GDNF fusion heavy chain, 70 kDa, is about 15 kDa larger than thesize of the heavy chain of the HIRMAb, owing to the fusion of the 15 kDaGDNF to the 55 kDa HIRMAb heavy chain

FIG. 32 . Binding of either the COS cell-derived chimeric HIRMAb or theHIRMAb-GDNF fusion protein to the HIR extracellular domain (ECD) issaturable. The ED50 of HIRMAb-GDNF binding to the HIR ECD is comparableto the ED50 of the binding of the chimeric HIRMAb.

FIG. 33 . (A) Outline of GFRα1 receptor binding assay. The GFRα1:Fcfusion protein is captured by a mouse anti-human (MAH) Fc antibody. TheGDNF, or HIRMAb-GDNF fusion protein, binds to the GFRα1, and thisbinding is detected with a goat anti-GDNF antibody and a rabbitanti-goat (RAG) antibody conjugated to alkaline phosphatase (AP). (B)Binding of either the COS cell derived chimeric HIRMAb or theHIRMAb-GDNF fusion protein to the GFRα1 extracellular domain (ECD) issaturable. The ED50 of HIRMAb-GDNF binding to the GFRα1 ECD iscomparable to the ED50 of the binding of recombinant GDNF. There is nobinding to the GFRα1 by the chimeric HIRMAb.

FIG. 34 . (A) Binding by either GDNF or the HIRMAb-GDNF fusion proteinto the GFRα1 on the cell membrane of c-ret kinase transfected humanneural SK-N-MC cells activates the tyrosine hydroxylase (TH) 5′-flankingsequence (FS), which drives luciferase gene expression. (B) Both GDNFand the COS cell-derived HIRMAb-GDNF fusion protein activate luciferasegene expression in a saturable manner, with ED50 values comparable toGFRα1 binding (FIG. 33B). There is no activation of luciferase geneexpression by the chimeric HIRMAb.

FIG. 35 . (A) Effect of intra-cerebral injection of the HIRMAb-GDNFversus saline on stroke volume in rats. Serial coronal sections of ratbrain at 24 hours after permanent MCAO and intra-cerebral saline (B) orHIRMAb-GDNF fusion protein (C) are stained with 2%2,3,5-triphenyltetrazolium chloride (TTC). A representative stain isshown for 1 rat from each treatment group. In these inverted grayscaleimages, the infarcted brain is black, and the healthy brain is white.Most rostral section is top and most caudal section is bottom.

FIG. 36 . Domain structure and amino acid sequence of the heavy chain(HC) of the HIRMAb-GDNF fusion protein (SEQ ID NO:46). The polypeptideis comprised of a signal peptide, followed by the variable region of theheavy chain (VH) of the chimeric HIRMAb. The 3 CDRs and 4 FRs are shown.The VH region is followed by the human IgG1 constant region, which iscomprised of the CH1, hinge, CH2, and CH3 domains, followed by a 2-aminoacid (Ser-Ser) linker, followed by the mature human GDNF. The singleN-linked glycosylation site within the CH2 region, and the 2 N-linkedglycosylation sites within the GDNF are underlined with the asparagine(N) residues in bold font. Cysteine (C) residues, which form inter-chaindisulfide bonds are shown, and include a linkage between the HC hingeregion and the light chain (LC), the HC hinge region and the paired HC,the HC CH2 region and the paired HC, and the GDNF-GDNF linkage. FIG. 36discloses the “signal peptide” sequence as residues 1-19 of SEQ ID NO:46, the “FR1” sequence as residues 20-44 of SEQ ID NO: 46, the “CDR1”sequence as residues 45-54 of SEQ ID NO: 46, the “FR2” sequence asresidues 55-68 of SEQ ID NO: 46, the “CDR2” sequence as residues 69-85of SEQ ID NO: 46, the “FR3” sequence as residues 86-117 of SEQ ID NO:46, the “CDR3” sequence as residues 118-121 of SEQ ID NO: 46, the “FR4”sequence as residues 122-132 of SEQ ID NO: 46, the “CH1” sequence asresidues 133-230 of SEQ ID NO: 46, the “hinge” sequence as residues231-242 of SEQ ID NO: 46, the “CH2” sequence as residues 243-355 of SEQID NO: 46, the “CH3” sequence as residues 356-462 of SEQ ID NO: 46 andthe “GDNF” sequence as residues 463-598 of SEQ ID NO: 46.

FIG. 37 . Tandem vector (TV) encoding multiple genes on a single pieceof DNA. The GDNF cDNA, produced by PCR, is subcloned into the HpaI siteof the universal tandem vector (UTV) to generate the HIRMAb-GDNF TV,which allows for expression in CHO cells of the fusion heavy chain (HC)gene, the light chain (LC) gene, and the dihydrofolate reductase (DHFR)gene.

FIG. 38 . Domain structure of the light chain of the HIRMAb (SEQ IDNO:48). FIG. 38 discloses the “signal peptide” sequence as residues 1-20of SEQ ID NO: 48, the “FR1” sequence as residues 21-43 of SEQ ID NO: 48,the “CDR1” sequence as residues 44-54 of SEQ ID NO: 48, the “FR2”sequence as residues 55-69 of SEQ ID NO: 48, the “CDR2” sequence asresidues 70-76 of SEQ ID NO: 48, the “FR3” sequence as residues 77-108of SEQ ID NO: 48, the “CDR3” sequence as residues 109-117 of SEQ ID NO:48, the “FR4” sequence as residues 118-128 of SEQ ID NO: 48 and the“kappa” sequence as residues 129-234 of SEQ ID NO: 48.

FIG. 39 . Western blot with a primary antibody against either human IgG(A) or human GDNF (B). The CHO-derived chimeric HIRMAb and theCHO-derived HIRMAb-GDNF fusion protein are applied in panel A, and theHIRMAb, the HIRMAb-GDNF fusion protein, and GDNF are applied in panel B.The migration of molecular weight standards is shown in lane 1 of panelA.

FIG. 40 . The affinity of the CHO-derived HIRMAb and the CHO-derivedHIRMAb-GDNF fusion protein for binding to the extracellular domain ofthe human insulin receptor (HIR) is measured in this ELISA format. Theconcentration of antibody that causes 50% saturation of binding, theED50, was determined by non-linear regression analysis. The 2 proteinshave the same affinity for the HIR, indicating fusion of GDNF to theC-terminus of the heavy chain of the HIRMAb does not affect binding tothe HIR.

FIG. 41 . Size exclusion high performance liquid chromatography of theHIRMAb-GDNF fusion protein shows the absence of aggregates in theCHO-derived protein.

FIG. 42 . Native polyacrylamide gel electrophoresis of the HIRMAb,control human IgG1, or the HIRMAb-GDNF fusion protein shows the absenceof aggregates in the CHO-derived fusion protein.

FIG. 43 . (A) Light micrograph of isolated human brain capillaries, usedas an in vitro model system of the human BBB. (B) Binding of the³H-HIRMAb-GDNF fusion protein to the isolated brain capillaries isinhibited by the murine HIRMAb. The component of HIRMAb-GDNF uptake bythe capillaries that is resistant to HIRMAb inhibition representsendocytosed fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

II. Definitions

III. The blood brain barrier

-   -   A. Transport systems    -   B. Structures that bind to a blood brain barrier        receptor-mediated transport system

IV. Agents for transport across the blood brain barrier

-   -   A. Neurotrophins    -   B. Brain-derived neurotrophic factor    -   C. Glial-derived neutrotrophic factor

V. Compositions

VI. Nucleic acids, vectors, cells, and manufacture

-   -   A. Nucleic acids    -   B. Vectors    -   C. Cells    -   D. Manufacture

VII. Methods

VIII. Kits

ABBREVIATIONS

-   -   AA amino acid    -   ALS amyotrophic lateral sclerosis    -   AP alkaline phosphatase    -   BBB blood-brain barrier    -   BCA bicinchoninic acid    -   BDNF brain derived neurotrophic factor    -   BGH bovine growth hormone    -   B max dose causing maximal effect    -   BSA bovine serum albumin    -   C cysteine    -   CDR compementarity determining region    -   CED convection enhanced diffusion    -   CHO Chinese hamster ovary    -   CMV cytomegalovirus    -   CNTF ciliary neurotrophic factor    -   DC dilutional cloning    -   DHFR dihydrofolate reductase    -   ECD extracellular domain    -   ED50 effective dose causing 50% saturation    -   FR framework region    -   FS flanking sequence    -   FWD forward    -   GDNF glial derived neurotrophic factor    -   GFR GDNF receptor    -   HC heavy chain    -   HIR human insulin receptor    -   HIRMAb MAb to HIR    -   HIRMAb-GDNF fusion protein of HIRMAb and GDNF    -   HPLC high pressure liquid chromatography    -   HT hypoxanthine-thymidine    -   ICV intra-cerebroventricular    -   ID injected dose    -   IgG immunoglobulin G    -   LC light chain    -   MAb monoclonal antibody    -   MAH mouse anti-human IgG    -   MCAO middle cerebral artery occlusion    -   MTX methotrexate    -   MW molecular weight    -   N asparagine    -   nt nucleotide    -   ODN oligodeoxynucleotide    -   pA poly-adenylation    -   PAGE polyacrylamide gel electrophoresis    -   PBS phosphate buffered saline    -   PBST PBS plus Tween-20    -   PCR polymerase chain reaction    -   PD Parkinson's disease    -   pI isoelectric point    -   RAG rabbit anti-goat IgG    -   REV reverse    -   RNase A ribonuclease A    -   RT reverse transcriptase    -   RT room temperature    -   SDM site-directed mutagenesis    -   SDS sodium dodecyl sulfate    -   SEC size exclusion chromatography    -   Ser serine    -   SFM serum free medium    -   SMA spinal muscular atrophy    -   TH tyrosine hydroxylase    -   TTC triphenyltetrazolium chloride    -   TV tandem vector    -   UTV universal TV    -   VH variable region of heavy chain    -   VL variable region of light chain

I. Introduction

The blood brain barrier is a limiting factor in the delivery of manyperipherally-administered agents to the central nervous system. Thepresent invention addresses three factors that are important indelivering an agent across the BBB to the CNS: 1) a pharmacokineticprofile for the agent that allows sufficient time in the peripheralcirculation for the agent to have enough contact with the BBB totraverse it; 2) modification of the agent to allow it to cross the BBB;and 3) retention of activity of the agent once across the BBB. Variousaspects of the invention address these factors, by providing fusionstructures (e.g., fusion proteins) of an agent (e.g., a therapeuticagent) covalently linked to a structure that causes the agent to haveincreased serum half life, to be transported across the BBB, and/or toretain some or all of its activity in the brain while still attached tothe structure.

Accordingly, in one aspect, the invention provides compositions andmethods that utilize an agent covalently linked to a structure capableof crossing the blood brain barrier (BBB). The compositions and methodsare useful in transporting agents, e.g. therapeutic agents such asneurotherapeutic agents, from the peripheral blood and across the BBBinto the CNS. Neurotherapeutic agents useful in the invention includeneurotrophins, e.g. brain-derived neurotrophic factor (BDNF) andglial-derived neurotrophic factor (GDNF). In some embodiments, thestructure that is capable of crossing the BBB is capable of binding toan endogenous BBB receptor mediated transport system and crossing theBBB. In some embodiments, the structure that is capable of crossing theBBB is an antibody, e.g., a monoclonal antibody (MAb) such as a chimericMAb.

In some embodiments, the invention provides a fusion protein thatincludes a structure capable of crossing the BBB covalently linked to apeptide that is active in the central nervous system (CNS), where thestructure capable of crossing the blood brain barrier and the peptidethat is active in the central nervous system each retain a proportion(e.g., 10-100%) of their activities or their binding affinities fortheir respective receptors, compared to their activities or theirbinding affinities for their respective receptors as separate entities.

In another aspect, the invention provides a composition containing acationic therapeutic peptide covalently linked to an immunoglobulin,where the cationic therapeutic peptide in the composition has a serumand/or circulating half-life that is an average of at least about fivefold greater than the serum and/or circulating half-life of the cationictherapeutic peptide alone.

The invention also provides nucleic acids coding for peptides andproteins. In some embodiments, the invention provides a single nucleicacid sequence that contains a gene coding for a light chain of animmunoglobulin and a gene coding for a fusion protein made up of a heavychain of the immunoglobulin covalently linked to a peptide. In someembodiments the peptide of the fusion protein is a therapeutic peptide,e.g., a neurotherapeutic peptide such as a neurotrophin. The inventionalso provides vectors containing the nucleic acids of the invention, andcells containing the vectors. Further provided are methods ofmanufacturing an immunoglobulin fusion protein, where the fusion proteincontains an immunoglobulin heavy chain fused to a therapeutic agent,where the methods include integrating into a eukaryotic cell a singletandem expression vector in which both the immunoglobulin light chaingene and the gene for the immunoglobulin heavy chain fused to thetherapeutic agent are incorporated into a single piece of DNA.

The invention further provides therapeutic compositions, such aspharmaceutical compositions that contain an agent covalently linked to astructure capable of crossing the blood brain barrier (BBB) and apharmaceutically acceptable excipient. In some embodiments, theinvention provides a composition for treating a neurological disorderthat includes a BDNF or a GDNF (e g , human GDNF) covalently linked toan immunoglobulin that is capable of crossing the blood brain barrier,wherein the composition is capable of crossing the BBB in an amount thatis effective in treating the neurological disorder.

The invention also provides methods for treating a neurological disorderin an individual that include peripherally administering to theindividual an effective amount of one or more of the compositions of theinvention, optionally in combination with other therapy for thedisorder.

II. Definitions

As used herein, an “agent” includes any substance that is useful inproducing an effect, including a physiological or biochemical effect inan organism. A “therapeutic agent” is a substance that produces or isintended to produce a therapeutic effect, i.e., an effect that leads toamelioration, prevention, and/or complete or partial cure of a disorder.A “therapeutic effect,” as that term is used herein, also includes theproduction of a condition that is better than the average or normalcondition in an individual that is not suffering from a disorder, i.e.,a supranormal effect such as improved cognition, memory, mood, or othercharacteristic attributable at least in part to the functioning of theCNS, compared to the normal or average state. A “neurotherapeutic agent”is an agent that produces a therapeutic effect in the CNS. A“therapeutic peptide” includes therapeutic agents that consists of apeptide. A “cationic therapeutic peptide” encompasses therapeuticpeptides whose isoelectric point is above about 7.4, in someembodiments, above about 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, orabove about 12.5. A subcategory of cationic therapeutic peptides iscationic neurotherapeutic peptides.

As used herein, a “peptide that is active in the central nervous system(CNS)” includes peptides that have an effect when administered to theCNS. The effect may be a therapeutic effect or a non-therapeutic effect,e.g., a diagnostic effect or an effect useful in research. If the effectis a therapeutic effect, then the peptide is also a therapeutic peptide.A therapeutic peptide that is also a peptide that is active in the CNSis encompassed by the term “neurotherapeutic peptide,” as used herein.

“Treatment” or “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderor condition being treated. For example, in an individual with aneurological disorder, therapeutic benefit includes partial or completehalting of the progression of the disorder, or partial or completereversal of the disorder. Also, a therapeutic benefit is achieved withthe eradication or amelioration of one or more of the physiological orpsychological symptoms associated with the underlying condition suchthat an improvement is observed in the patient, notwithstanding the factthat the patient may still be affected by the condition. A prophylacticbenefit of treatment includes prevention of a condition, retarding theprogress of a condition (e.g., slowing the progression of a neurologicaldisorder), or decreasing the likelihood of occurrence of a condition. Asused herein, “treating” or “treatment” includes prophylaxis.

As used herein, the term “effective amount” can be an amount sufficientto effect beneficial or desired results, such as beneficial or desiredclinical results, or enhanced cognition, memory, mood, or other desiredCNS results. An effective amount is also an amount that produces aprophylactic effect, e.g., an amount that delays, reduces, or eliminatesthe appearance of a pathological or undesired condition. Such conditionsof the CNS include dementia, neurodegenerative diseases as describedherein, suboptimal memory or cognition, mood disorders, general CNSaging, or other undesirable conditions. An effective amount can beadministered in one or more administrations. In terms of treatment, an“effective amount” of a composition of the invention is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of a disorder, e.g., a neurological disorder. An “effectiveamount” may be of any of the compositions of the invention used alone orin conjunction with one or more agents used to treat a disease ordisorder. An “effective amount” of a therapeutic agent within themeaning of the present invention will be determined by a patient'sattending physician or veterinarian. Such amounts are readilyascertained by one of ordinary skill in the art and will a therapeuticeffect when administered in accordance with the present invention.Factors which influence what a therapeutically effective amount will beinclude, the specific activity of the therapeutic agent being used, thetype of disorder (e.g., acute vs. chronic neurological disorder), timeelapsed since the initiation of the disorder, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe individual being treated. Additionally, other medication the patientmay be receiving will affect the determination of the therapeuticallyeffective amount of the therapeutic agent to administer

A “subject” or an “individual,” as used herein, is an animal, forexample, a mammal In some embodiments a “subject” or an “individual” isa human In some embodiments, the subject suffers from a neurologicaldisorder.

In some embodiments, an agent is “administered peripherally” or“peripherally administered.” As used herein, these terms refer to anyform of administration of an agent, e.g., a therapeutic agent, to anindividual that is not direct administration to the CNS, i.e., thatbrings the agent in contact with the non-brain side of the blood-brainbarrier. “Peripheral administration,” as used herein, includesintravenous, subcutaneous, intramuscular, intraperitoneal, transdermal,inhalation, transbuccal, intranasal, rectal, and oral administration.

A “pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” herein refers to any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Such carriers are well known to those of ordinary skill inthe art. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA.Exemplary pharmaceutically acceptable carriers can include salts, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. Forexample, compositions of the invention may be provided in liquid form,and formulated in saline based aqueous solution of varying pH (5-8),with or without detergents such polysorbate-80 at 0.01-1%, orcarbohydrate additives, such mannitol, sorbitol, or trehalose. Commonlyused buffers include histidine, acetate, phosphate, or citrate.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof, including, but notlimited to, degenerate codon substitutions, and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid may be one that isseparated from the nucleic acids that normally flank it or other nucleicacids or components (proteins, lipids, etc. . . . ) in a sample. Inanother example, a polypeptide is purified if it is substantiallyremoved from or concentrated in its natural environment. Methods forpurification and isolation of nucleic acids and peptides are well knownin the art.

III. The Blood Brain Barrier

In one aspect, the invention provides compositions and methods thatutilize an agent covalently linked to a structure capable of crossingthe blood brain barrier (BBB). The compositions and methods are usefulin transporting agents, e.g. therapeutic agents such as neurotherapeuticagents, from the peripheral blood and across the BBB into the CNS. Asused herein, the “blood-brain barrier” refers to the barrier between theperipheral circulation and the brain and spinal cord which is formed bytight junctions within the brain capillary endothelial plasma membranes,creates an extremely tight barrier that restricts the transport ofmolecules into the brain, even molecules as small as urea, molecularweight of 60 Da. The blood-brain barrier within the brain, theblood-spinal cord barrier within the spinal cord, and the blood-retinalbarrier within the retina, are contiguous capillary barriers within thecentral nervous system (CNS), and are collectively referred to as theblood-brain barrier or BBB.

The BBB is a limiting step in the development of new neurotherapeutics,diagnostics, and research tools for the brain and CNS. In general, largemolecule therapeutics such as recombinant proteins, antisense drugs,gene medicines, monoclonal antibodies, or RNA interference (RNAi)-baseddrugs, do not cross the BBB in pharmacologically significant amounts.While it is generally assumed that small molecule drugs can cross theBBB, in fact, <2% of all small molecule drugs are active in the brainowing to the lack transport across the BBB. A molecule must be lipidsoluble and have a molecular weight less than 400 Daltons (Da) in orderto cross the BBB in pharmacologically significant amounts, and the vastmajority of small molecules do not have these dual molecularcharacteristics. Therefore, most potentially therapeutic, diagnostic, orresearch molecules do not cross the BBB in pharmacologically activeamounts. So as to bypass the BBB, invasive transcranial drug deliverystrategies are used, such as intracerebro-ventricular (ICV) infusion,intracerebral (IC) administration, and convection enhanced diffusion(CED). Transcranial drug delivery to the brain is expensive, invasive,and largely ineffective. The ICV route delivers BDNF only to theependymal surface of the brain, not into brain parenchyma, which istypical for drugs given by the ICV route. The IC administration of aneurotrophin, such as nerve growth factor (NGF), only delivers drug tothe local injection site, owing to the low efficiency of drug diffusionwithin the brain. The CED of neurotrophin results in preferential fluidflow through the white matter tracts of brain, which causesdemyelination, and astrogliosis.

The present invention offers an alternative to these highly invasive andgenerally unsatisfactory methods for bypassing the BBB, allowing agents,e.g., neuroprotective factors to cross the BBB from the peripheralblood. It is based on the use of endogenous transport systems present inthe BBB to provide a mechanism to transport a desired substance from theperipheral blood to the CNS.

A. Transport Systems

In some embodiments, the invention provides compositions that include astructure that binds to a BBB receptor mediated transport system,coupled to an agent for which transport across the BBB is desired, e.g.,a neurotherapeutic agent. The compositions and methods of the inventionmay utilize any suitable structure that is capable of transport by theselected endogenous BBB receptor-mediated transport system, and that isalso capable of attachment to the desired agent. In some embodiments,the structure is an antibody. In some embodiment the antibody is amonoclonal antibody (MAb), e.g., a chimeric MAb.

Endogenous BBB receptor-mediated transport systems The BBB has beenshown to have specific receptors that allow the transport from the bloodto the brain of several macromolecules; these transporters are suitableas transporters for compositions of the invention. Endogenous BBBreceptor-mediated transport systems useful in the invention includethose that transport insulin, transferrin, insulin-like growth factors 1and 2 (IGF1 and IGF2), leptin, and lipoproteins. In some embodiments,the invention utilizes a structure that is capable of crossing the BBBvia the endogenous insulin BBB receptor-mediated transport system, e.g.,the human endogenous insulin BBB receptor-mediated transport system.

B. Structures that Bind to a BBB Receptor Mediated Transport System

One noninvasive approach for the delivery of drugs to the CNS is toattach the agent of interest to a structure, e.g., molecule that bindswith receptors on the BBB. The structure then serves as a vector fortransport of the agent across the BBB. Such structures are referred toherein as “molecular Trojan horses (MTH).” Typically, though notnecessarily, a MTH is an exogenous peptide or peptidomimetic moiety(e.g., a MAb) capable of binding to an endogenous BBB receptor mediatedtransport system that traverses the BBB on the endogenous BBBreceptor-mediated transport system. In certain embodiments, the MTH canbe an antibody to a receptor of the transport system, e.g., the insulinreceptor. In some embodiments, the antibody is a monoclonal antibody(MAb). In some embodiments, the MAb is a chimeric MAb. Thus, despite thefact that Abs normally are excluded from the brain, they can be aneffective vehicle for the delivery of molecules into the brainparenchyma if they have specificity for receptors on the BBB.

Accordingly, antibodies are particularly useful in embodiments of theinvention, especially MAbs. Certain receptor-specific MAbs may mimic theendogenous ligand and function as a MTH and traverse a plasma membranebarrier via transport on the specific receptor system. In certainembodiments, the MTH is a MAb to the human insulin receptor (HIR) on thehuman BBB. The HIR MAb binds an exofacial epitope on the human BBB HIRand this binding enables the MAb to traverse the BBB via a transportreaction that is mediated by the human BBB insulin receptor.

An “antibody,” as that term is used herein, includes reference to anymolecule, whether naturally-occurring, artificially induced, orrecombinant, which has specific immunoreactive activity. Generally,though not necessarily, an antibody is a protein that includes twomolecules, each molecule having two different polypeptides, the shorterof which functions as the light chains of the antibody and the longer ofwhich polypeptides function as the heavy chains of the antibody.Normally, as used herein, an antibody will include at least one variableregion from a heavy or light chain. Additionally, the antibody maycomprise combinations of variable regions. The combination may includemore than one variable region of a light chain or of a heavy chain Theantibody may also include variable regions from one or more light chainsin combination with variable regions of one or more heavy chains Anantibody can be an immunoglobulin molecule obtained by in vitro or invivo generation of the humoral response, and includes both polyclonaland monoclonal antibodies. Furthermore, the present invention includesantigen binding fragments of the antibodies described herein, such asFab, Fab′, F(ab)₂, and Fv fragments, fragments comprised of one or moreCDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)),disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies(e.g., bispecific antibodies), pFv fragments, heavy chain monomers ordimers, light chain monomers or dimers, and dimers consisting of oneheavy chain and one light chain Such antibody fragments may be producedby chemical methods, e.g., by cleaving an intact antibody with aprotease, such as pepsin or papain, or via recombinant DNA techniques,e.g., by using host cells transformed with truncated heavy and/or lightchain genes. Synthetic methods of generating such fragments are alsocontemplated. Heavy and light chain monomers may similarly be producedby treating an intact antibody with a reducing agent, such asdithiothreitol or beta-mercaptoethanol, or by using host cellstransformed with DNA encoding either the desired heavy chain or lightchain or both. An antibody immunologically reactive with a particularantigen can be generated in vivo or by recombinant methods such asselection of libraries of recombinant antibodies in phage or similarvectors.

A “chimeric” antibody includes an antibody derived from a combination ofdifferent mammals The mammal may be, for example, a rabbit, a mouse, arat, a goat, or a human. The combination of different mammals includescombinations of fragments from human and mouse sources.

In some embodiments, an antibody of the present invention is amonoclonal antibody (MAb), typically a human monoclonal antibody. Suchantibodies are obtained from transgenic mice that have been “engineered”to produce specific human antibodies in response to antigenic challenge.In this technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas.

For use in humans, a chimeric MAb is preferred that contains enoughhuman sequence that it is not significantly immunogenic whenadministered to humans, e g , at least about 80% human and about 20%mouse, or about 85% human and about 15% mouse, or about 90% human andabout 10% mouse, or about 95% human and 5% mouse, or greater than about95% human and less than about 5% mouse. Chimeric antibodies to the humanBBB insulin receptor with sufficient human sequences for use in theinvention are described in, e.g., Coloma et al. (2000) Pharm. Res. 17:266-274, which is incorporated by reference herein in its entirety. Amore highly humanized form of the HIR MAb can also be engineered, andthe humanized HIRMAb has activity comparable to the murine HIRMAb andcan be used in embodiments of the invention. See, e.g., U.S. PatentApplication Publication No. 20040101904, filed Nov. 27, 2002,incorporated by reference herein in its entirety.

Antibodies used in the invention may be glycosylated ornon-glycosylated. If the antibody is glycosylated, any pattern ofglycosylation that does not significantly affect the function of theantibody may be used. Glycosylation can occur in the pattern typical ofthe cell in which the antibody is made, and may vary from cell type tocell type. For example, the glycosylation pattern of a monoclonalantibody produced by a mouse myeloma cell can be different than theglycosylation pattern of a monoclonal antibody produced by a transfectedChinese hamster ovary (CHO) cell. In some embodiments, the antibody isglycosylated in the pattern produced by a transfected Chinese hamsterovary (CHO) cell.

Accordingly, in some embodiments, a genetically engineered HIR MAb, withthe desired level of human sequences, is fused to an agent for whichtransport across the BBB is desired, e.g., a neurotherapeutic agent suchas a neurotrophin such as human BDNF, to produce a recombinant fusionprotein that is a bi-functional molecule. The recombinant therapeuticneuroprotective factor/HIRMAb is able to both (i) cross the human BBB,via transport on the BBB HIR, and (ii) activate the factor's target,e.g., neuronal BDNF receptor, trkB, to cause neurotherapeutic effectsonce inside the brain, following peripheral administration.

IV. Agents for Transport Across the BBB

The agent for which transport across the BBB is desired may be anysuitable substance for introduction into the CNS. Generally, the agentis a substance for which transport across the BBB is desired, which doesnot, in its native form, cross the BBB in significant amounts. The agentmay be, e.g., a therapeutic agent, a diagnostic agent, or a researchagent. Diagnostic agents include peptide radiopharmaceuticals, such asradiolabeled epidermal growth factor (EGF) for imaging brain cancer(Kurihara and Pardridge (1999) Canc. Res. 54: 6159-6163), and amyloidpeptides for imaging brain amyloid such as in Alzheimers disease (Lee etal (2002) J. Cereb. Blood Flow Metabol. 22: 223-231). In someembodiments, the agent is a therapeutic agent, such as aneurotherapeutic agent. Apart from neurotrophins, potentially usefultherapeutic protein agents include recombinant enzymes for lysosomalstorage disorders (see, e.g., U.S. Patent Application Publication No.20050142141, filed Feb. 17, 2005, incorporated by reference herein inits entirety), monoclonal antibodies that either mimic an endogenouspeptide or block the action of an endogenous peptide, polypeptides forbrain disorders, such as secretin for autism (Ratliff-Schaub et al(2005) Autism 9: 256-265), opioid peptides for drug or alchol addiction(Cowen et al, (2004) J. Neurochem. 89: 273-285), or neuropeptides forappetite control (Jethwa et al (2005) Am. J. Physiol. 289: E301-305). Insome embodiments, the agent is a neurotrophic factor, also referred toherein as a “neurotrophin.” Thus, in some embodiments, the inventionprovides compositions and methods that utilize a neurotrophin. In someembodiments, a single neurotrophin may be used. In others, combinationsof neurotrophins are used. In some embodiments, the invention utilizes abrain-derived neurotrophic factor (BDNF). In other embodiments, theinvention utilizes a glial-derived neurotrophic factor.

A. Neurotrophins

Many neurotrophic factors are neuroprotective in brain, but do not crossthe blood-brain barrier. These factors are suitable for use in thecompositions and methods of the invention and include brain-derivedneurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5,fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-1 receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, and stem cell factor (SCF). Particularly usefulin some embodiments of the invention utilizing neurotrophins that areused as precursors for fusion proteins that cross the BBB are those thatnaturally form dimeric structures, similar to BDNF. Certainneurotrophins such as BDNF or NT-3 may form hetero-dimeric structures,and in some embodiments the invention provides a fusion proteinconstructed of one neurotrophin monomer fused to one chain (e.g., alight or heavy chain) of an antibody, e.g., of the HIRMAb, and anotherneurotrophin monomer fused to the second chain (e.g., a light or heavychain) of the antibody. Typically, the molecular weight range ofrecombinant proteins that may be fused to the molecular Trojan horseranges from 1000 Daltons to 500,000 Daltons.

B. Brain-Derived Neurotrophic Factor

One particularly useful neurotrophin in embodiments of the invention isbrain-derived neurotrophic factor (BDNF). In experimental models ofchronic neurodegenerative disease such as prion diseases, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD), oramyotrophic lateral sclerosis (ALS), the direct intracerebral injectionof BDNF is neuroprotective.

In studies demonstrating the pharmacologic efficacy of BDNF inexperimental brain disease, it is necessary to administer theneurotrophin directly into the brain following a transcranial drugdelivery procedure. The transcranial drug delivery is required becauseBDNF does not cross the brain capillary wall, which forms theblood-brain barrier (BBB) in vivo. Owing to the lack of transport ofBDNF across the BBB, it is not possible for the neurotrophin to enterthe CNS, including the brain or spinal cord, following a peripheraladministration unless the BBB is experimentally disrupted. Clinicaltrials showed that subcutaneous administration of BDNF was not effectivein the treatment of chronic neurodegenerative conditions, which derivesfrom the lack of transport of BDNF across the BBB. The lack of utilityof BDNF as a CNS therapeutic following peripheral administration isexpected and follows from the limiting role that is played by the BBB inthe development of neurotherapeutics, especially large molecule drugssuch as BDNF. BDNF does not cross the BBB, and the lack of transport ofthe neurotrophin across the BBB prevents the molecule from beingpharmacologically active in the brain following peripheraladministration. The lack of BDNF transport across the BBB means that theneurotrophin must be directly injected into the brain across the skullbone to be pharmacologically active in the CNS. However, when the BDNFis fused to a Trojan horse such as the HIR MAb, this neurotrophin is nowable to enter brain from blood following a non-invasive peripheral routeof administration such as intravenous intramuscular, subcutaneous,intraperitoneal, or even oral administration. Owing to the BBB transportproperties of this new class of molecule, it is not necessary toadminister the BDNF directly into the CNS with an invasive deliveryprocedure requiring penetration of the skull or spinal canal. Thereformulated fusion protein of the BDNF variant and the HIR MAb nowenables entry of BDNF into the brain from the blood, and the developmentof BDNF as a neurotherapeutic for human diseases.

The forms of BDNF used in various embodiments of the invention mayinclude pharmaceutically acceptable salts and prodrugs, and prodrugs ofthe salts, polymorphs, hydrates, solvates, biologically-activefragments, biologically active variants and stereoisomers of thenaturally-occurring BDNF, as well as agonist, mimetic, and antagonistvariants of the naturally-occurring BDNF and polypeptide fusionsthereof. Variants that include one or more deletions, substitutions, orinsertions in the natural sequence of the BDNF, in particular truncatedversions of the native BDNF comprising deletion of one or more aminoacids at the amino terminus, carboxyl terminus, or both, may also beused in certain embodiments.

In some embodiments, the invention utilizes a carboxy-truncated variantof the native BDNF, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10 amino acids are absent from the carboxy-terminus ofthe BDNF. BDNF variants include the complete 119 amino acid BDNF, the117 or 118 amino acid variant with a truncated carboxyl terminus,variants with a truncated amino terminus, or variants with up to about a20, 30, or 40% change in amino acid composition, as long as the fusionprotein variant still binds to the brain neuroprotection receptor withhigh affinity. When an Ab, e.g., a MAb such as HIRMAb is used,additional fusion protein variants can be produced with the substitutionof amino acids within either the framework region (FR) or thecomplementarity determining region (CDR) of either the light chain orthe heavy chain of the Ab, e.g., HIRMAb, as long as the fusion proteinbinds with high affinity to the endogenous receptor, e.g., HIR topromote transport across the human BBB. Additional fusion proteinvariants can be produced by changing the composition or length of thelinker peptide separating the fusion protein from the HIRMAb.

In some embodiments, the full-length 119 a.a. sequence of BDNF isutilized (SEQ ID NO: 39). In some embodiments, a one amino-acidcarboxy-truncated variant of BDNF is utilized (amino acids 1-118 of SEQID NO: 39). In some embodiments, a two amino-acid carboxy-truncatedvariant of BDNF is utilized (amino acids 1-117 of SEQ ID NO: 39). Insome embodiments, a three amino-acid carboxy-truncated variant of BDNFis utilized (amino acids 1-116 of SEQ ID NO: 39). In some embodiments, afour amino-acid carboxy-truncated variant of BDNF is utilized (aminoacids 1-115 of SEQ ID NO: 39). In some embodiments, a five amino-acidcarboxy-truncated variant of BDNF is utilized (amino acids 1-114 of SEQID NO: 39).

The sequence of human BDNF is given in SEQ ID NO: 39. In someembodiments, the invention utilizes a BDNF that is about 60, 70, 80, 90,95, 99, or 100% identical with the sequence of SEQ ID NO: 39, or atruncated version thereof, e.g., the 117 or 118 amino acid variant witha one- or two-amino acid truncated carboxyl terminus, or variants with atruncated amino terminus. In some embodiments, the invention utilizes atwo amino-acid carboxy-truncated 117 amino acid variant human BDNF witha sequence that is at least about 60, 70, 80, 90, 95, 99 or 100%identical to the sequence of amino acids 466-582 of SEQ ID NO: 24. Insome embodiments, the invention utilizes a two amino-acidcarboxy-truncated human 117 amino acid BDNF with a sequence thatincludes amino acids 466-582 of SEQ ID NO: 24.

Accordingly, BDNFs useful in the invention include peptides having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 99%, or greater than 95% orgreater than 99% sequence identity, e.g., 100% sequence identity, to theamino acid sequences disclosed herein.

C. Glial-Derived Neurotrophic Factor.

Another particularly useful neurotrophin in embodiments of the inventionis glial-derived neurotrophic factor (GDNF). GDNF is a neurotrophicfactor that can be used in the treatment of many acute and chronic braindiseases. However, the lack of transport of GDNF across the BBB hasprevented the development of this molecule as a neurotherapeutic for thebrain and spinal cord.

Acute brain conditions that can be treated by the GDNF-containingcompositions described herein include, but are not limited to, spinalcord injury, brain injury (e.g., traumatic brain injury), focal brainischemia, and global brain ischemia.

Chronic brain conditions that can be treated by the GDNF-containingcompositions described herein include, but are not limited toneurodegenerative disease such as Parkinson's disease, motor neurondisease (e.g., spinal cord motor neuron disease such as amyotrophiclateral sclerosis); and substance abuse, e.g., addiction, including drugaddiction and alchohol addiction.

The forms of GDNF used in various embodiments of the invention mayinclude acceptable salts and prodrugs, and prodrugs of the salts,polymorphs, hydrates, solvates, biologically-active fragments,biologically active variants and stereoisomers of thenaturally-occurring GDNF (e g , human GDNF and mature human GDNF), aswell as agonist, mimetic, and antagonist variants of thenaturally-occurring GDNF and polypeptide fusions thereof. Variants thatinclude one or more deletions, substitutions, or insertions in thenatural sequence of the GDNF may also be used in certain embodiments.insofar as the variants retain binding and/or activation of GFRα1. Thestructure-function relationship of GDNF and its ability to bind andactivate GFRα1 has been studied extensively. See, e.g., Eketjäl et al(1999), EMBO J, 18(21):5901-5910; and Baloh et al (2000), J Biol Chem,275(5):3412-3420. For example mutations known to be particularlysensitive to mutation include, e.g., the following residues in rat GDNF:D52, E61, E62, D116, I64, L114, L118, Y120, I122, and C101. Functionalassays for GDNF are known in the art. See, e.g., Eketjäl et al, or Balohet al supra. Functional assays for GDNF are also described in Example 15herein.

When an Ab, e.g., a MAb such as HIRMAb is used, additional fusionprotein variants can be produced with the substitution of amino acidswithin either the framework region (FR) or the complementaritydetermining region (CDR) of either the light chain or the heavy chain ofthe Ab, e.g., HIRMAb, as long as the fusion protein binds with highaffinity to the endogenous receptor, e.g., HIR to promote transportacross the human BBB. Additional fusion protein variants can be producedby changing the composition or length of the linker peptide separatingthe fusion protein from the HIRMAb.

In some embodiments, the full-length 211 a.a. sequence of human preproGDNF is utilized (GenBank P39905). The sequence of human preproGDNF isgiven in SEQ ID NO: 44. In other embodiments, the mature form of humanGDNF comprising amino acids Ser-78 to Ile 211 (SEQ ID NO: 52) isutilized:SPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCG CI(SEQ ID NO:52).

In some embodiments, the invention utilizes a GDNF that is about 60, 70,80, 90, 95, 99, or 100% identical with the sequence of SEQ ID NO:52.

Accordingly, GDNFs useful in the invention include peptides having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 99%, or greater than 95% orgreater than 99% sequence identity, e.g., 100% sequence identity, to theamino acid sequences disclosed herein

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of anotherpeptide. The FASTA algorithm is described by Pearson and Lipman, Proc.Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:44 orSEQ ID NO:52) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

The present invention also includes peptides having a conservative aminoacid change, compared with an amino acid sequence disclosed herein. Manysuch changes have been described specifically. More generally, forexample, variants can be obtained that contain one or more amino acidsubstitutions of SEQ ID NO:52. In some embodiments sequence variantsinclude conservative amino acid substitutions, e.g., an alkyl amino acidis substituted for an alkyl amino acid in a GDNF peptide amino acidsequence, an aromatic amino acid is substituted for an aromatic aminoacid in a GDNF peptide amino acid sequence, a sulfur-containing aminoacid is substituted for a sulfur-containing amino acid in a GDNF peptideamino acid sequence, a hydroxy-containing amino acid is substituted fora hydroxy-containing amino acid in a GDNF peptide amino acid sequence,an acidic amino acid is substituted for an acidic amino acid in a GDNFpeptide amino acid sequence, a basic amino acid is substituted for abasic amino acid in GDNF peptide amino acid sequence, or a dibasicmonocarboxylic amino acid is substituted for a dibasic monocarboxylicamino acid in a GDNF peptide amino acid sequence. Among the common aminoacids, for example, a “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine. The BLOSUM62 table is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff and Henikoff, Proc.Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62substitution frequencies can be used to define conservative amino acidsubstitutions that may be introduced into the amino acid sequences ofthe present invention. Although it is possible to design amino acidsubstitutions based solely upon chemical properties (as discussedabove), the language “conservative amino acid substitution” preferablyrefers to a substitution represented by a BLOSUM62 value of greater than−1. For example, an amino acid substitution is conservative if thesubstitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

It also will be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids,and yet still be essentially as set forth in one of the sequencesdisclosed herein, so long as the sequence retains sufficient biologicalprotein activity to be functional in the compositions and methods of theinvention.

In some embodiments, where GDNF sequence variants (e.g., variants of SEQID NO:52) are to be utilized, mutation tolerance prediction programs canbe used to greatly reduce the number of non-functional sequence variantsthat would be generated by strictly random mutagenesis. Various programsfor predicting the effects of amino acid substitutions in a proteinsequence on protein function (e.g., SIFT, PolyPhen, PANTHER PSEC, PMUT,and TopoSNP) are described in, e.g., Henikoff et al., (2006), Annu. Rev.Genomics Hum. Genet., 7:61-80.

D. Alpha-L-Iduronidase

Alpha-L-iduronidase (IDUA) is the enzyme missing in patients with Hurlersyndrome or type I mucopolysaccharidosis (MPS), which adversely affectsthe brain. The brain pathology ultimately results in early death forchildren carrying this genetic disease. IDUA enzyme replacement therapy(ERT) for patients with MPS type I is not effective for the braindisease, because the enzyme does not cross the BBB. This is a seriousproblem and means the children with this disease will die early eventhough they are on ERT. The enzyme could be delivered across the humanBBB following peripheral administration providing the enzyme is attachedto a molecular Trojan horse such as the humanized HIRMAb. The IDUA maybe attached to the humanized HIRMAb with avidin-biotin technology. Inthis approach, the IDUA enzyme is mono-biotinylated in parallel with theproduction of a fusion protein of the humanized HIRMAb and avidin. Inaddition, the IDUA could be attached to the humanized HIRMAb not withavidin-biotin technology, but with genetic engineering that avoids theneed for biotinylation or the use of foreign proteins such as avidin. Inthis approach, the gene encoding for IDUA is fused to the region of thehumanized HIRMAb heavy chain or light chain gene corresponding to theamino or carboxyl terminus of the HIRMAb heavy or light chain protein.

V. Compositions

Compositions of the invention are useful in one or more of: increasingserum half-life of a cationic compound, transporting an agent across theBBB, and/or retaining activity of the agent once transported across theBBB. Accordingly, in some embodiments, the invention providescompositions containing a neurotherapeutic agent covalently linked to astructure that is capable of crossing the blood brain barrier (BBB),where the composition is capable of producing an average elevation ofconcentration in the brain of the neurotherapeutic agent of at leastabout 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 ng/gram brain followingperipheral administration. The invention also provides compositionscontaining an agent that is covalently linked to a chimeric MAb to thehuman BBB insulin receptor. The invention further provides a fusionprotein containing a structure capable of crossing the BBB, covalentlylinked to a peptide that is active in the central nervous system (CNS),where the structure capable of crossing the blood brain barrier and thepeptide that is active in the central nervous system each retain anaverage of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of their activities, compared to their activities as separateentities. In certain embodiments, the invention further providescompositions that increase the serum half-life of cationic substances.The invention also provides pharmaceutical compositions that contain oneor more compositions of the invention and a pharmaceutically acceptableexcipient.

In some embodiments, the invention provides compositions containing aneurotherapeutic agent covalently linked to a structure that is capableof crossing the blood brain barrier (BBB), where the composition iscapable of producing an average elevation of concentration in the brainof the neurotherapeutic agent of at least about 1, 2, 3, 4, 5, 10, 20,30, 40, or 50 ng/gram brain following peripheral administration.

“Elevation” of the agent is an increase in the brain concentration ofthe agent compared to the concentration of the agent administered alone(i.e., not covalently linked to a structure that is capable of crossingthe BBB). In the case of agents for which only a small amount of theagent alone normally crosses the BBB, “elevation” may be an increase inthe agent compared to resting brain levels. “Average” refers to the meanof at least three, four, five, or more than five measurements,preferably in different individuals. The individual in which theelevation is measured is a mammal, such as a rat, or, preferably, aprimate, e.g., a monkey. An example of measurements of elevation of thelevel of a neurotherapeutic agent (BDNF) is given in Example 7.

In some embodiments, the structure that is capable of crossing the BBButilizes an endogenous BBB receptor mediated transport system, such as asystem that utilizes the insulin receptor, transferrin receptor, leptinreceptor, LDL receptor, or IGF receptor. In some embodiments, theendogenous BBB receptor mediated transport system is the insulin BBBreceptor mediated transport system. In some embodiments, the structurethat is capable of crossing the BBB is an antibody, e.g., a monoclonalantibody (MAb) such as a chimeric MAb. The antibody can be a chimericantibody with sufficient human sequence that it is suitable foradministration to a human. The antibody can be glycosylated ornonglycosylated; in some embodiments, the antibody is glycosylated,e.g., in a glycosylation pattern produced by its synthesis in a CHOcell. In embodiments in which the structure is an antibody, the covalentlinkage between the antibody and the neurotherapeutic agent may be alinkage between any suitable portion of the antibody and theneurothempeutic agent, as long as it allows the antibody-agent fusion tocross the blood brain barrier and the neurotherapeutic agent to retain atherapeutically useful portion of its activity within the CNS. Incertain embodiments, the covalent link is between one or more lightchains of the antibody and the neurotherapeutic agent. In the case of apeptide neurotherapeutic agent (e.g., a neurotrophin such as GDNF), thepeptide can be covalently linked by its carboxy or amino terminus to thecarboxy or amino terminus of the light chain (LC) or heavy chain (HC) ofthe antibody. Any suitable linkage may be used, e.g., carboxy terminusof light chain to amino terminus of peptide, carboxy terminus of heavychain to amino terminus of peptide, amino terminus of light chain toamino terminus of peptide, amino terminus of heavy chain to aminoterminus of peptide, carboxy terminus of light chain to carboxy terminusof peptide, carboxy terminus of heavy chain to carboxy terminus ofpeptide, amino terminus of light chain to carboxy terminus of peptide,or amino terminus of heavy chain to carboxy terminus of peptide. In someembodiments, the linkage is from the carboxy terminus of the HC to theamino terminus of the peptide. It will be appreciated that a linkagebetween terminal amino acids is not required, and any linkage whichmeets the requirements of the invention may be used; such linkagesbetween non-terminal amino acids of peptides are readily accomplished bythose of skill in the art.

In some embodiments, the invention utilizes BDNF, either the native formor truncated variants. Strikingly, it has been found that fusionproteins of these forms of BDNF retain full transport and activity. Thisis surprising because the neurotrophin is translated in vivo in cells asa prepro form and the prepro-BDNF is then converted into mature BDNFfollowing cleavage of the prepro peptide from the amino terminus of theBDNF. In order to preserve the prepro form of the BDNF, and thesubsequent cleavability of the prepro peptide, it would seem to benecessary to fuse the prepro BDNF to the amino terminus of either the HCor the LC of the targeting MAb. This could, however, inhibit the bindingof the MAb for the target antigen, since the complementarity determiningregions (CDR) of the heavy chain or light chain of the MAb molecule,which comprise the antigen binding site of the MAb, are situated nearthe amino terminus of the heavy chain or light chains of the antibody.Therefore, fusion of the prepro-neurotrophin to the amino terminus ofthe antibody chains is expected to result in not only impairment ofantibody activity, but also an impairment of antibody folding followingtranslation. The present invention shows the unexpected finding that itis possible to fuse the mature form of a neurotrophin, such as a BDNFvariant (vBDNF), to the carboxyl terminus of the heavy chain of the HIRMAb. The production of this new genetically engineered fusion proteincreates a bi-functional molecule that binds with high affinity to boththe HIR and the trkB receptors.

In other embodiments, the invention utilizes GDNF (e.g., mature humanGDNF) or a sequence variant of GDNF as described herein.

In some embodiments, more than one molecule of the same neurotherapeuticagent is attached to the structure that crosses the BBB. For example, incompositions of the invention where a single neurotrophin is attached toan antibody, one molecule of the neurotrophin is attached to each heavychain, naturally producing a structure that is ideal for homodimerformation. This is the case for compositions containing BDNF or GDNF.Neurotrophins such as BDNF or GDNF require an obligatory formation of ahomo-dimeric structure to be biologically active, and to bind with highaffinity to the cognate receptor, e.g. TrkB or GFRα1. A naturallyoccurring homo-dimeric structure between two BDNF molecules is formedwhen the neurotrophin is fused to a carboxyl terminus of the CH3 regionof an IgG molecule, as illustrated in FIG. 18 . Without being bound bytheory, it is thought that this may account for the unexpected findingof essentially 100% of activity for the BDNF when bound to the IgG (see,e.g., FIG. 24 ).

In some embodiments, more than one type of neurotherapeutic agent can beattached to the structure that is capable of crossing the blood brainbarrier. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10 different neurotherapeutic agents may be attached to the structurethat is capable of crossing the blood brain barrier. In certainembodiments, 2 different neurotrophins are attached to an antibody to anendogenous BBB receptor-mediated transport system. Any combination ofneurotrophins may be used. Particularly useful in some embodiments ofthe invention are neurotrophins used as precursors for fusion proteinsthat cross the BBB are those that naturally form dimeric structures,similar to BDNF or GDNF. Certain neurotrophins such as BDNF or NT-3 mayform hetero-dimeric structures, and in some embodiments the inventionprovides a fusion protein constructed of one neurotrophin monomer fusedto one chain (e.g., heavy chain) of an antibody, e.g., of the HIRMAb,and another neurotrophin monomer fused to the second chain of theantibody. Typically, the molecular weight range of recombinant proteinsthat may be fused to the molecular Trojan horse ranges from 1000 Daltonsto 500,000 Daltons.

In some embodiments, more than one type of structure capable of crossingthe BBB, e.g., molecular Trojan horse, may be used. The differentstructures may be covalently attached to a single neurotherapeuticagent, e.g., a single neurotrophin such as GDNF, or multipleneurotherapeutics, e.g., multiple neurotrophins, or any combinationthereof. Thus, for example, in some embodiments either with the sameneurotrophin attached to each MTH or a different neurotrophin attached,or combinations of neurotrophins attached. Thus the neuroprotectiverecombinant protein can be fused to multiple molecular Trojan horsesthat undergo receptor-mediated transport across the blood-brain barrier,including monoclonal antibodies to the insulin receptor, transferrinreceptor, insulin-like growth factor (IGF) receptor, or the low densitylipoprotein (LDL) receptor or the endogenous ligand, including insulin,transferrin, the IGFs, or LDL. Ligands that traverse the blood-brainbarrier via absorptive-mediated transport may also be used as molecularTrojan horses including cationic proteins, or carbohydrate bearingproteins that bind to membrane lectins. The molecular weight range ofmolecular Trojan horses is 1000 Daltons to 500,000 Daltons.

The covalent linkage between the structure capable of crossing the BBBand the neurotherapeutic agent may be direct, e.g., a peptide bondbetween the terminal amino acid of one peptide and the terminal aminoacid of the other peptide to which it is linked, or indirect, via alinker. If a linker is used, it may be any suitable linker, e.g., apeptide linker. If a peptide linker is used, it may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10 amino acids in length. In some embodiments,a three amino acid linker is used. In some embodiments, the linker hasthe sequence ser-ser-met. The covalent linkage may be cleavable, howeverthis is not a requirement for activity of the system in someembodiments; indeed, an advantage of these embodiments of the presentinvention is that the fusion protein, without cleavage, is partially orfully active both for transport and for activity once across the BBB.

In some embodiments, a noncovalent attachment may be used. An example ofnoncovalent attachment of the MTH, e.g., MAb, to the large moleculetherapeutic neuroprotective factor is avidin/streptavidin-biotinattachment. Such an approach is further described in U.S. patentapplication Ser. No. 10/858,729, entitled “Anti-growth factor receptoravidin fusion proteins as universal vectors for drug delivery,” filedApr. 21, 2005, which is hereby incorporated by reference in itsentirety.

The neurotherapeutic agent may be any suitable neurothempeutic agent,such as a neurotrophin. In some embodiments, the neurotherapeutic agentis a neurotrophin such as brain derived neurotrophic factor (BDNF),nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor(FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO),hepatocyte growth factor (HGF), epidermal growth factor (EGF),transforming growth factor (TGF)-α, TGF-β, vascular endothelial growthfactor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliaryneurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF),neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin,artemin, persephin, interleukins, granulocyte-colony stimulating factor(CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF. TheBDNF may be native BDNF or a variant BDNF. Some embodiments utilize atwo amino acid carboxyl-truncated variant. The BDNF can be a human BDNF.In some embodiments, the BDNF contains a sequence that is about 60, 70,80, 90, 95, 99, or 100% identical to the sequence of amino acids 466-582of SEQ ID NO: 24.

In some embodiments, the invention provides compositions containing aneurotherapeutic agent covalently linked to a structure that is capableof crossing the BBB where the composition is capable of producing anaverage elevation of concentration in the brain of the neurotherapeuticagent of at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 ng/grambrain following peripheral administration, where the neurotherapeuticagent is a neurotrophin and the structure that is capable of crossingthe BBB is a MAb to an endogenous BBB receptor mediated transportsystem. The antibody can be glycosylated or nonglycosylated; in someembodiments, the antibody is glycosylated, e.g., in a glycosylationpattern produced by its synthesis in a CHO cell. In certain embodiments,the neurotrophin is GDNF, e.g., a mature human GDNF (SEQ ID NO:52) or asequence variant thereof. The MAb can be an antibody to the insulin BBBreceptor mediated transport system, e.g., a chimeric MAb. The antibodycan be a chimeric antibody with sufficient human sequence that it issuitable for administration to a human, e.g., at least about 80% humansequence, e.g., 85%, 90%, 95%, or another percent human amino acidsequence from about 80% to about 100% human sequence. In someembodiments, the insulin receptor is a human insulin receptor and theGDNF is a human GDNF. In some embodiments, the GDNF contains a sequencethat is about 60, 70, 80, 90, 95, 99, or 100% identical to the sequenceof SEQ ID NO: 44. The GDNF can be covalently linked at its aminoterminus to the carboxy terminus of the heavy chain of the MAb,optionally with a linker between the termini, such as the threeamino-acid linker ser-ser-met, or the two amino acid linker ser-ser. Insome embodiments, the heavy chain of the MAb contains a sequence that isabout 60, 70, 80, 90, 95, 99, or 100% identical to amino acids 20-462 ofSEQ ID NO: 24. In some embodiments, the light chain of the MAb containsa sequence that is about 60, 70, 80, 90, 95, 99, or 100% identical toamino acids 21-234 of SEQ ID NO: 36.

The invention also provides compositions containing an agent that iscovalently linked to a chimeric MAb to the human BBB insulin receptor.In some embodiments, the heavy chain of the MAb is covalently linked tothe agent to form a fusion protein. The agent can be any agent describedherein, i.e., any agent for which transport across the BBB is desired.In some embodiments, the agent is a therapeutic agent, such as aneurotherapeutic agent as described herein, e.g., a neurotrophin such asGDNF

Strikingly, it has been found that multifunctional fusion proteins ofthe invention, e.g., difunctional fusion proteins, retain a highproportion of the activity of the separate portions, e.g., the portionthat is capable of crossing the BBB and the portion that is active inthe CNS. Accordingly, the invention further provides a fusion proteincontaining a structure capable of crossing the BBB, covalently linked toa peptide that is active in the central nervous system (CNS), where thestructure capable of crossing the BBB and the peptide that is active inthe central nervous system each retain an average of at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activities,compared to their activities as separate entities. In some embodiments,the structure capapble of crossing the BBB, and the peptide that isactive in the central nervous system each retain about 20% to about 80%of their activities (e.g., about 30% to about 70, or about 40% to about60%) compared to their activities as separate entities. In someembodiments, the invention provides a fusion protein containing astructure capable of crossing the BBB, covalently linked to a peptidethat is active in the central nervous system (CNS), where the structurecapable of crossing the blood brain barrier and the peptide that isactive in the central nervous system each retain an average of at leastabout 50% of their activities, compared to their activities as separateentities. In some embodiments, the invention provides a fusion proteincontaining a structure capable of crossing the BBB, covalently linked toa peptide that is active in the central nervous system (CNS), where thestructure capable of crossing the blood brain barrier and the peptidethat is active in the central nervous system each retain an average ofat least about 60% of their activities, compared to their activities asseparate entities. In some embodiments, the invention provides a fusionprotein containing a structure capable of crossing the BBB, covalentlylinked to a peptide that is active in the central nervous system (CNS),where the structure capable of crossing the blood brain barrier and thepeptide that is active in the central nervous system each retain anaverage of at least about 70% of their activities, compared to theiractivities as separate entities. In some embodiments, the inventionprovides a fusion protein containing a structure capable of crossing theBBB, covalently linked to a peptide that is active in the centralnervous system (CNS), where the structure capable of crossing the bloodbrain barrier and the peptide that is active in the central nervoussystem each retain an average of at least about 80% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a fusion protein containing a structure capableof crossing the BBB, covalently linked to a peptide that is active inthe central nervous system (CNS), where the structure capable ofcrossing the blood brain barrier and the peptide that is active in thecentral nervous system each retain an average of at least about 90% oftheir activities, compared to their activities as separate entities. Insome embodiments, the structure capable of crossing the blood brainbarrier retains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95,99, or 100% of its activity, compared to its activity as a separateentity, and the peptide that is active in the central nervous systemretains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of its activity, compared to its activity as a separate entity.

As used herein, “activity” includes physiological activity, e.g.,ability to cross the BBB and/or therapeutic activity and also bindingaffinity of the structures for their respective receptors.

Transport of the structure capable of crossing the BBB across the BBBmay be compared for the structure alone and for the structure as part ofa fusion structure of the invention by standard methods. For example,pharmacokinetics and brain uptake of the fusion structure, e.g., fusionprotein, by a model animal, e g , a mammal such as a primate, may beused. Such techniques are illustrated in Example 7, which demonstratespharmacokinetics and brain uptake of a fusion protein of the inventionby the adult Rhesus monkey. Similarly, standard models for the functionof an agent, e.g. the therapeutic or protective function of atherapeutic agent, may also be used to compare the function of the agentalone and the function of the agent as part of a fusion structure of theinvention. See, e.g., Example 5, which demonstrates the activity of aneurotrophin alone and the same neurotrophin bound to a fusion proteinin a model system (hypoxia-reoxygenation in human neural cells). In bothExample 5 and Example 7, the fusion protein of the invention retainedabout 100% of the transport ability and the therapeutic function of itsindividual components, i.e., a structure capable of crossing the BBB (aMAb to the human insulin receptor) and a therapeutic agent (BDNF).

Alternatively, binding affinity for receptors may be used as a marker ofactivity. Binding affinity for the receptor is compared for thestructure alone and for the structure when part of the fusion protein. Asuitable type of binding affinity assay is the competitive ligandbinding assay (CLBA). For example, for fusion proteins containing MAbsto endogenous BBB receptor-mediated transport systems fused to aneurotrophin, a CLBA may be used both to assay the affinity of the MAbfor its receptor and the neurotrophin for its receptor, either as partof the fusion protein or as separate entities, and percentage affinitycalculated. If, as in some embodiments, the peptide that is active inthe CNS is highly ionic, e.g., cationic, causing a high degree ofnon-specific binding, suitable measures should be taken to eliminate thenonspecific binding. See, e.g., Example 4. “Average” measurements arethe average of at least three separate measurements.

In embodiments of the above fusion proteins, the structure capable ofcrossing the blood brain barrier crosses the BBB on an endogenous BBBreceptor-mediated transporter, such as a transporter selected from thegroup consisting of the insulin transporter, the transferrintransporter, the leptin transporter, the LDL transporter, and the IGFreceptor. In some embodiments, the endogenous BBB receptor-mediatedtransporter is selected from the group consisting of the insulintransporter and the transferrin transporter. In some embodiments, theendogenous BBB receptor-mediated transporter is the insulin transporter,e.g., the human insulin transporter. The structure capable of crossingthe BBB can be an antibody, e.g., a MAb such as a chimeric MAb. Theantibody can be an antibody to an endogenous BBB receptor-mediatedtransporter, as described herein. The peptide that is active in the CNScan be a neurotherapeutic agent, e.g., a neurotrophin. In someembodiments, the neurotrophin is selected from the group consisting ofbrain-derived neurotrophic factor, nerve growth factor (NGF),neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs,neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor(HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF such asa truncated BDNF, e.g., a carboxyl-truncated BDNF. Thecarboxyl-truncated BDNF is lacking the two carboxyl terminal amino acidsin some embodiments. The structure capable of crossing the BBB and theneurotherapeutic agent are covalently linked by a peptide linker in someembodiments.

In certain embodiments, the invention provides compositions thatincrease the serum half-life of cationic substances. One limitation formany current therapeutics, especially cationic therapeutic peptides(e.g., BDNF) is their rapid clearance from the circulation. The positivecharge on the cationic substance, such as cationic peptides, rapidlyinteracts with negative charges on cell membranes, which triggers anabsorptive-mediated endocytosis into the cell, particularly liver andspleen. This is true not only for neurotherapeutics (where rapidclearance means only limited contact with the BBB and thus limitedability to cross the BBB) but for other agents as well, such as cationicimport peptides such as the tat peptide, or cationic proteins (e.g.protamine, polylysine, polyarginine) that bind nucleic acids, orcationic proteins such as avidin that bind biotinylated drugs.Surprisingly, fusion compositions of the invention that include acationic therapeutic peptide covalently linked to an immunoglobulin showgreatly enhanced serum half-life compared to the same peptide when itwas not covalently part of a fusion immunoglobulin. This is an importantfinding, because it shows that the fusion of a highly cationic protein,e.g., BDNF, to an immunoglobulin, e.g. HIRMAb, has two important andunexpected effects: 1) it greatly enhances the serum half-life of thecationic protein, and 2) it does not accelerate the blood clearance ofthe immunoglobulin to which it is attached, e.g., the HIRMAb. Prior workshows that the noncovalent attachment of a cationic therapeutic peptide,e.g., the cationic BDNF to a monoclonal antibody greatly accelerated theblood clearance of the antibody, owing to the cationic nature of theBDNF, which greatly enhances hepatic uptake. The work in FIG. 27A andExample 7 shows that when the cationic therapeutic peptide, e.g., BDNFis re-engineered as an IgG fusion protein, the plasma pharmacokineticsis dominated by the IgG moiety, and that the blood level of the BDNFremains high for a prolonged period; indeed, the serum half-life of theBDNF in the fusion protein is at least about 100 times that of the BDNFalone.

Accordingly, in some embodiments, the invention provides compositioncomprising a cationic therapeutic peptide covalently linked to animmunoglobulin, wherein the cationic therapeutic peptide in thecomposition has a serum half-life that is an average of at least about1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, or more than about 100-fold greater than the serum half-life of thecationic therapeutic peptide alone. In some embodiments, the inventionprovides a composition comprising a cationic therapeutic peptidecovalently linked to an immunoglobulin, wherein the cationic therapeuticpeptide in the composition has a mean residence time (MRT) in the serumthat is an average of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than about 100-foldgreater than the serum half-life of the cationic therapeutic peptidealone. In some embodiments, the invention provides compositioncomprising a cationic therapeutic peptide covalently linked to animmunoglobulin, wherein the cationic therapeutic peptide in thecomposition has a systemic clearance rate that is an average of at leastabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, or more than about 100-fold slower than the systemic clearancerate of the cationic therapeutic peptide alone. In some embodiments, theinvention provides composition comprising a cationic therapeutic peptidecovalently linked to an immunoglobulin, wherein the cationic therapeuticpeptide in the composition has average blood level after peripheraladministration that is an average of at least about 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than about100-fold greater than the average blood level after peripheraladministration of the cationic therapeutic peptide alone.

In some embodiments, the cationic therapeutic peptide comprises aneurotherapeutic agent. Examples of neurotherapeutic agents that arecationic peptides interferons, interleukins, cytokines, or growthfactors with an isoelectric point (pI) above 8. In some embodiments, theneurotherapeutic agent is a neurotrophin. Cationic peptide neurotrophinsinclude BDNF, GDNF, NT-3, NT-4/5, NGF, and FGF-2. In some embodiments,the neurotrophin is BDNF or GDNF.

In some embodiments, the immunoglobulin is an antibody to an endogenousBBB receptor-mediated transport system. In some embodiments, theendogenous BBB receptor-mediated transport system is selected from thegroup consisting of the insulin BBB transport system, the BBBtransferrin receptor, the BBB leptin receptor, the BBB IGF receptor, orthe BBB lipoprotein receptor. In some embodiments, the antibody is anantibody to the endogenous insulin BBB receptor-mediated transportsystem. Antibodies can be any suitable antibody as described herein.

Pharmaceutical compositions The invention also provides pharmaceuticalcompositions that contain one or more compositions of the invention anda pharmaceutically acceptable excipient. A thorough discussion ofpharmaceutically acceptable carriers/excipients can be found inRemington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition,2000: Williams and Wilkins PA, USA. Pharmaceutical compostions of theinvention include compositions suitable for administration via anyperipheral route, including intravenous, subcutaneous, intrmuscular,intraperitoneal injection; oral, rectal, transbuccal, pulmonary,transdermal, intranasal, or any other suitable route of peripheraladministration.

The compostions of the invention are particular suited for injection,e.g., as a pharmaceutical composition for intravenous, subcutaneous,intramuscular, or intraperitonal administration. Aqueous compositions ofthe present invention comprise an effective amount of a composition ofthe present invention, which may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, e g , a human,as appropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Exemplary pharmaceutically acceptable carriers for injectablecompositions can include salts, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. For example, compositions of the invention maybe provided in liquid form, and formulated in saline based aqueoussolution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol; phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate, and gelatin

For human administration, preparations meet sterility, pyrogenicity,general safety, and purity standards as required by FDA and otherregulatory agency standards The active compounds will generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition thatcontains an active component or ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use in preparing solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation include vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed

The term “unit dose” refers to physically discrete units suitable foruse in a subject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. The person responsible for administration will, inany event, determine the appropriate dose for the individual subject.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1 0 milligrams, or about 0.001 to 0.1milligrams, or about 1.0 to 100 milligrams or even about 0.01 to 1.0grams per dose or so. Multiple doses can also be administered. In someembodiments, a dosage of about 2.5 to about 25 mg of a fusion protein ofthe invention is used as a unit dose for administration to a human, e g, about 2.5 to about 25 mg of a fusion protein of GDNF and a HIR MAb.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other alternativemethods of administration of the present invention may also be used,including but not limited to intradermal administration (See U.S. Pat.Nos. 5,997,501; 5,848,991; and 5,527,288), pulmonary administration (SeeU.S. Pat. Nos. 6,361,760; 6,060,069; and 6,041,775), buccaladministration (See U.S. Pat. Nos. 6,375,975; and 6,284,262),transdermal administration (See U.S. Pat. Nos. 6,348,210; and 6,322,808)and transmucosal administration (See U.S. Pat. No. 5,656,284). All suchmethods of administration are well known in the art. One may also useintranasal administration of the present invention, such as with nasalsolutions or sprays, aerosols or inhalants. Nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions. Thus, the aqueous nasal solutionsusually are isotonic and slightly buffered to maintain a pH of 5.5 to6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

Additional formulations, which are suitable for other modes ofadministration, include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. For suppositories, traditional bindersand carriers generally include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in any suitable range, e.g., in the range of 0.5%to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in a hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations can contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried, and may conveniently be between about 2 to about 75% of theweight of the unit, or between about 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent, methylene and propyl parabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Insome embodiments, an oral pharmaceutical composition may be entericallycoated to protect the active ingredients from the environment of thestomach; enteric coating methods and formulations are well-known in theart.

VI. Nucleic Acids, Vectors, Cells, and Manufacture.

The invention also provides nucleic acids, vectors, cells, and methodsof production.

A. Nucleic Acids

In some embodiments, the invention provides nucleic acids that code forproteins or peptides of the invention. In certain embodiments, theinvention provides a single nucleic acid sequence containing a firstsequence coding for a light chain of an immunoglobulin and secondsequence coding a heavy chain of the immunoglobulin, where either thefirst sequence also codes for a peptide that is expressed as a fusionprotein of the peptide covalently linked to the light chain, or thesecond sequence also codes for a peptide that is expressed as a fusionprotein of the peptide covalently linked to the heavy chain In someembodiments, the invention provides nucleic acid sequences, and in someembodiments the invention provides nucleic acid sequences that are atleast about 60, 70, 80, 90, 95, 99, or 100% identical to a particularnucleotide sequence. For example, in some embodiments, the inventionprovides a nucleic acid containing a first sequence that is at leastabout 60, 70, 80, 90, 95, 99, or 100% identical to SEQ ID NO:45and asecond sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to nucleotides 1396-1746 of SEQ ID NO: 33.

In other embodiments, the invention provides a nucleic acid containing asequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to SEQ ID NO:45.

For sequence comparison, of two nucleic acids, typically one sequenceacts as a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. The BLAST algorithm is typicallyperformed with the “low complexity” filter turned off. The BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The invention provides nucleic acids that code for any of the peptidesof the invention. In some embodiments, the invention provides a singlenucleic acid sequence containing a gene coding for a light chain of animmunoglobulin and a gene coding for a fusion protein, where the fusionprotein includes a heavy chain of the immunoglobulin covalently linkedto a peptide. In some embodiments, the peptide is a therapeutic peptide.In some embodiments the peptide is a neurothempeutic peptide, e.g., aneurotrophin such as BDNF or GDNF (e.g., mature human GDNF). In someembodiments, the BDNF is a two amino acid carboxy-truncated BDNF. Insome embodiments, the immunoglobulin is an IgG. In some embodiments, theIgG is a MAb, such as a chimeric MAb. The antibody can be an antibody toa transport system, e.g., an endogenous BBB receptor-mediated transportsystem such as the endogenous BBB receptor-mediated insulin transportsystem. In some embodiments, the endogenous BBB receptor-mediatedinsulin transport system is a human endogenous BBB receptor-mediatedinsulin transport system and wherein the peptide to which theimmunoglobulin heavy chain is covalently linked is human BDNF or humanGDNF. Any suitable peptide, neurotherapeutic peptide, neurotrophin,BDNF, GDNF (e.g., mature human GDNF), antibody, monoclonal antibody, orchimeric antibody, as described herein, may be coded for by the nucleicacid, combined as a fusion protein and coded for in a single nucleicacid sequence. As is well-known in the art, owing to the degeneracy ofthe genetic code, any combination of suitable codons may be used to codefor the desired fusion protein. In addition, other elements useful inrecombinant technology, such as promoters, termination signals, and thelike, may also be included in the nucleic acid sequence. Such elementsare well-known in the art. In addition, all nucleic acid sequencesdescribed and claimed herein include the complement of the sequence.

In some embodiments the nucleic acid codes for a BDNF, e.g., a variantBDNF, or GDNF (e.g., mature human GDNF) as a component of the fusionprotein, which also comprises an immunoglobulin sequence. In someembodiments, the BDNF contains a sequence that is about 60, 70,80, 90,95, 99, or 100% identical to the sequence of amino acids 466-582 of SEQID NO: 24. In some embodiments, the GDNF contains a sequence that is atleast about 60, 70,80, 90, 95, 99, or 100% identical to the sequence ofSEQ ID NO:52. In some embodiments, the amino acid sequence of theencoded GDNF consists essentially of SEQ ID NO:52. In some embodiments,the nucleic acid codes for a fusion protein comprising an amino acidsequence that is at least about 60, 70,80, 90, 95, 99, or 100% identicalto the sequence of SEQ ID NO:46. In some embodiments, the encodednucleic acid comprises the amino acid sequence of SEQ ID NO:46. In someembodiments, the BDNF or GDNF is linked at its amino terminus to carboxyterminus of the heavy chain of the immunoglobulin, e.g., MAb. The heavychain of the MAb can comprise a sequence that is about 60, 70, 80, 90,95, 99 or 100% identical to amino acids 20-462 of SEQ ID NO: 24. In someembodiments, the light chain of the immunoglobulin, e.g., MAb, comprisesa sequence that is about 60, 70, 80, 90, 95, 99 or 100% identical toamino acids 21-234 of SEQ ID NO: 36. The nucleic acid can furthercontain a nucleic acid sequence that codes for a peptide linker betweenthe heavy chain of the MAb and the BDNF or GDNF. In some embodiments,the linker is S-S-M. The nucleic acid may further contain a nucleic acidsequence coding for a signal peptide, wherein the signal peptide islinked to the heavy chain Any suitable signal peptide, as known in theart or subsequently developed, may be used. In some embodiments, thesignal peptide attached to the heavy chain comprises a sequence that isabout 60, 70, 80, 90, 95, 99, or 100% identical to amino acids 1-19 ofSEQ ID NO: 24. In some embodiments, the nucleic acid contains a nucleicacid sequence coding for another signal peptide, wherein the othersignal peptide is linked to the light chain The signal peptide linked tothe light chain can comprise a sequence that is about 60, 70, 80, 90,95, 99, or 100% identical to amino acids 1-20 of SEQ ID NO: 36. Thenucleic acid can contain a nucleic acid sequence coding for a selectablemarker. In some embodiments the selectable marker is DHFR. The sequenceof the DHFR can be about 60, 70, 80, 90, 95, 99, or 100% identical toamino acids 1-187 of SEQ ID NO: 38.

In certain embodiments, the invention provides a nucleic acid comprisinga first sequence that codes for a neurotherapeutic peptide, e.g., aneurotrophin such as BDNF, in the same open reading frame as a secondsequence that codes for an immunoglobulin component. The immunoglobulincomponent can be, e.g., a light chain or a heavy chain, e g , that is atleast about 60, 70, 80, 90, 95, 99, or 100% identical to nucleotides58-1386-of SEQ ID NO: 33 and a second sequence that is at least about60, 70, 80, 90, 95, 99, or 100% identical to nucleotides 1396-1746 ofSEQ ID NO: 33. In some embodiments, the nucleic acid also contains athird sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to nucleotides 61-702 of SEQ ID NO: 35. In some embodiments,the nucleic acid further contains a fourth sequence that codes for afirst signal peptide and a fifth sequence that codes for a second signalpeptide. In some embodiments, the fourth sequence is at least about 60,70, 80, 90, 95, 99, or 100% identical to nucleotides 1-57 of SEQ ID NO:33 and the fifth sequence is at least about 60, 70, 80, 90, 95, 99, or100% identical to nucleotides 1-60 of SEQ ID NO: 35. In someembodiments, the nucleic acid further contains a sequence that codes fora selectable marker, such as dihydrofolate reductase (DHFR). In someembodiments, the sequence that codes for the DHFR is at least about 60,70, 80, 90, 95, 99, or 100% identical to nucleotides 1-561 of SEQ ID NO:37.

B. Vectors

The invention also provides vectors. The vector can contain any of thenucleic acid sequences described herein. In some embodiments, theinvention provides a single tandem expression vector containing nucleicacid coding for an antibody heavy chain fused to a peptide, e.g., atherapeutic peptide such as a neurotrophin, and nucleic acid coding fora light chain of the antibody, all incorporated into a single piece ofnucleic acid, e.g., a single piece of DNA. The single tandem vector canalso include one or more selection and/or amplification genes. A methodof making an exemplary vector of the invention is provided in theExamples. However, any suitable techniques, as known in the art, may beused to construct the vector.

The use of a single tandem vector has several advantages over previoustechniques. The transfection of a eukaryotic cell line withimmunoglobulin G (IgG) genes generally involves the co-transfection ofthe cell line with separate plasmids encoding the heavy chain (HC) andthe light chain (LC) comprising the IgG. In the case of a IgG fusionprotein, the gene encoding the recombinant therapeutic protein may befused to either the HC or LC gene. However, this co-transfectionapproach makes it difficult to select a cell line that has equally highintegration of both the HC and LC-fusion genes, or the HC-fusion and LCgenes. The approach to manufacturing the fusion protein utilized incertain embodiments of the invention is the production of a cell linethat is stably transfected with a single plasmid DNA that contains allthe required genes on a single strand of DNA, including the HC-fusionprotein gene, the LC gene, the selection gene, e.g. neo, and theamplification gene, e.g. the dihydrofolate reductase gene. As shown inthe diagram of the fusion protein tandem vector in FIG. 12 , theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

C. Cells

The invention further provides cells that incorporate one or more of thevectors of the invention. The cell may be a prokaryotic cell or aeukaryotic cell. In some embodiments, the cell is a eukaryotic cell. Insome embodiments, the cell is a mouse myeloma hybridoma cell. In someembodiments, the cell is a Chinese hamster ovary (CHO) cell. Exemplarymethods for incorporation of the vector(s) into the cell are given inthe Examples. However, any suitable techniques, as known in the art, maybe used to incorporate the vector(s) into the cell. In some embodiments,the invention provides a cell capable of expressing an immunoglobulinfusion protein, where the cell is a cell into which has been introduceda single tandem expression vector, where both the immunoglobulin lightchain gene and the gene for the immunoglobulin heavy chain fused to thetherapeutic agent, are incorporated into a single piece of nucleic acid,e.g., DNA. In some embodiments, the invention provides a cell capable ofexpressing an immunoglobulin fusion protein, where the cell is a cellinto which has been stably transfected a single tandem expressionvector, where both the immunoglobulin heavy chain gene and the gene forthe immunoglobulin light chain fused to the therapeutic agent, areincorporated into a single piece of nucleic acid, e.g., DNA. Theintroduction of the tandem vector may be by, e.g., permanent integrationinto the chromsomal nucleic acid, or by, e.g., introduction of anepisomal genetic element.

D. Methods of Manufacture

In addition, the invention provides methods of manufacture. In someembodiments, the invention provides a method of manufacturing animmunoglobulin fusion protein, where the fusion protein contains animmunoglobulin heavy chain fused to a therapeutic agent, by introducinginto a eukaryotic cell a single tandem expression vector, where both theimmunoglobulin light chain gene and the gene for the immunoglobulinheavy chain fused to the therapeutic agent, are incorporated into asingle piece of nucleic acid, e.g., DNA. In some embodiments, theinvention provides a method of manufacturing an immunoglobulin fusionprotein, where the fusion protein contains an immunoglobulin light chainfused to a therapeutic agent, by ly introducing into a eukaryotic cell asingle tandem expression vector, where both the immunoglobulin heavychain gene and the gene for the immunoglobulin light chain fused to thetherapeutic agent, are incorporated into a single piece of nucleic acid,e.g., DNA. In some embodiments, the introduction of the vector isaccomplished by integration into the host cell genome. In someembodiments, the introduction of the vector is accomplished byintroduction of an episomal genetic element containing the vector intothe host cell. Episomal genetic elements are well-known in the art Insome embodiments, the therapeutic agent is a neurotherapeutic agent. Insome embodiments, the single piece of nucleic acid further includes oneor more genes for selectable markers. In some embodiments, the singlepiece of nucleic acid further includes one or more amplification genes.In some embodiments, the immunoglobulin is an IgG, e.g., a MAb such as achimeric MAb. The methods may further include expressing theimmunoglobulin fusion protein, and/or purifying the immunoglobulinfusion protein. Exemplary methods for manufacture, including expressionand purification, are given in the Examples.

However, any suitable techniques, as known in the art, may be used tomanufacture, optionally express, and purify the proteins. These includenon-recombinant techniques of protein synthesis, such as solid phasesynthesis, manual or automated, as first developed by Merrifield anddescribed by Stewart et al. in Solid Phase Peptide Synthesis (1984).Chemical synthesis joins the amino acids in the predetermined sequencestarting at the C-terminus. Basic solid phase methods require couplingthe C-terminal protected a-amino acid to a suitable insoluble resinsupport. Amino acids for synthesis require protection on the a-aminogroup to ensure proper peptide bond formation with the preceding residue(or resin support). Following completion of the condensation reaction atthe carboxyl end, the a-amino protecting group is removed to allow theaddition of the next residue. Several classes of α-protecting groupshave been described, see Stewart et al. in Solid Phase Peptide Synthesis(1984), with the acid labile, urethane-based tertiary-butyloxycarbonyl(Boc) being the historically preferred. Other protecting groups, and therelated chemical strategies, may be used, including the base labile9-fluorenylmethyloxycarbonyl (FMOC). Also, the reactive amino acidsidechain functional groups require blocking until the synthesis iscompleted. The complex array of functional blocking groups, along withstrategies and limitations to their use, have been reviewed by Bodanskyin Peptide Synthesis (1976) and, Stewart et al. in Solid Phase PeptideSynthesis (1984).

Solid phase synthesis is initiated by the coupling of the describedC-terminal a-protected amino acid residue. Coupling requires activatingagents, such as dicyclohexycarbodiimide (DCC) with or without1-hydroxybenzo-triazole (HOBT), diisopropylcarbodiimide (DIIPC), orethyldimethylaminopropylcarbodiimide (EDC). After coupling theC-terminal residue, the a-amino protected group is removed bytrifluoroacetic acid (25% or greater) in dichloromethane in the case ofacid labile tertiary-butyloxycarbonyl (Boc) groups. A neutralizing stepwith triethylamine (10%) in dichloro-methane recovers the free amine(versus the salt). After the C-terminal residue is added to the resin,the cycle of deprotection, neutralization and coupling, withintermediate wash steps, is repeated in order to extend the protectedpeptide chain Each protected amino acid is introduced in excess (threeto five fold) with equimolar amounts of coupling reagent in suitablesolvent. Finally, after the completely blocked peptide is assembled onthe resin support, reagents are applied to cleave the peptide form theresin and to remove the side chain blocking groups. Anhydrous hydrogenfluoride (HF) cleaves the acid labile tertiary-butyloxycathonyl (Boc)chemistry groups. Several nucleophilic scavengers, such asdimethylsulfide and anisole, are included to avoid side reactionsespecially on side chain functional groups.

VII. Methods

The invention also provides methods. In some embodiments, the inventionprovides methods for transport of an agent active in the CNS across theBBB in an effective amount. In some embodiments, the invention providestherapeutic, diagnostic, or research methods. Diagnostic methods includethe development of peptide radiopharmaceuticals capable of transportacross the BBB, such as the fusion of a peptide ligand, orpeptidomimetic MAb for an endogenous receptor in the brain, followed bythe radiolabelling of the fusion protein, followed by systemicadministration, and external imaging of the localization within thebrain of the peptide radiopharmaceutical.

Prior to the present invention, neurotrophins such as BDNF or GDNF wereinjected directly into the brain to achieve a therapeutic effect,because the neurotrophin does not cross the BBB. Therefore, it is notexpected that neurotrophic factors will have beneficial effects on braindisorders following the peripheral (intravenous, subcutaneous)administration of these molecules.

However, neurotherapeutics can be developed as drugs for peripheralroutes of administration, providing the neurotherapeutic is enabled tocross the BBB. Attachment of the neurotherapeutic, e.g. a neurotrophinsuch as BDNF or GDNF to a MTH, e.g., the chimeric HIRMAb, offers a newapproach to the non-invasive delivery of neurotherapeutics to the CNS inanimals, e g , mammals such as humans for the treatment of acute brainand spinal cord conditions, such as focal brain ischemia, global brainischemia, and spinal cord injury, and chronic treatment ofneurodegenerative disease, including prion diseases, Alzheimer's disease(AD), Parkinson's disease (PD), Huntington's disease (HD), ALS, multiplesclerosis, transverse myelitis, motor neuron disease, Pick's disease,addiction (e.g., drug addiction), tuberous sclerosis, lysosomal storagedisorders, Canavan's disease, Rett's syndrome, spinocerebellar ataxias,Friedreich's ataxia, optic atrophy, and retinal degeneration.

Accordingly, in some embodiments the invention provides methods oftransport of an agent active in the CNS from the peripheral circulationacross the BBB in an effective amount, where the agent is covalentlyattached to a structure that crosses the BBB, and where the agent aloneis not transported across the BBB in an effective amount. In someembodiments the invention provides methods of transport ofneurotherapeutic agent from the peripheral circulation across the BBB ina therapeutically effective amount, where the neurotherapeutic agent iscovalently attached to a structure that crosses the BBB, and where theneurotherapeutic agent alone is not transported across the BBB in atherapeutically effective amount.

The invention also provides, in some embodiments, methods of treatmentof disorders of the CNS by peripheral administration of an effectiveamount of a therapeutic agent, e.g., a neurotherapeutic agent covalentlylinked to a structure that is capable of crossing the BBB, where theagent alone is not capable of crossing the BBB in an effective amountwhen administered peripherally. In some embodiments, the CNS disorder isan acute disorder, and, in some cases, may require only a singleadministration of the agent. In some embodiments, the CNS disorder is achronic disorder and may require more than one administration of theagent.

In some embodiments, the effective amount, e.g., therapeuticallyeffective amount is such that a concentration in the brain is reached ofat least about 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 100, or more than 100 ng/gram brain. In someembodiments, a therapeutically effective amount, e.g., of a neurotrophinsuch as BDNF or GDNF, is such that a brain level is achieved of about0.1 to 1000, or about 1-100, or about 5-50 ng/g brain. In someembodiments, the neurotherapeutic agent is a neurotrophin. In someembodiments, the neurotrophin is selected from the group consisting ofBDNF, nerve growth factor (NGF), neurotrophin-4/5, fibroblast growthfactor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin(EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF),transforming growth factor (TGF)-α, TGF-β, vascular endothelial growthfactor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliaryneurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF),neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin,artemin, persephin, interleukins, granulocyte-colony stimulating factor(CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF, e.g. atruncated BDNF, such as the carboxyl-truncated BDNFs described herein.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a neurotrophin, where theneurotrophin is capable of crossing the BBB to produce an averageelevation of neurotrophin concentration in the brain of at least about0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100,or more than 100 ng/gram brain following said peripheral administration,and where the neurotrophin remains at the elevated level for about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10 days after a singleadministration. In some embodiments, the neurotrophin remains at a levelof greater than about 1 ng/g brain, or about 2 ng/g brain, or about 5ng/g brain for about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 daysafter a single administration. In some embodiments, the neurotrophin isBDNF, including truncated versions thereof.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a composition of theinvention. The term “peripheral administration,” as used herein,includes any method of administration that is not direct administrationinto the CNS, i.e., that does not involve physical penetration ordisruption of the BBB. “Peripheral administration” includes, but is notlimited to, intravenous intramuscular, subcutaneous, intraperitoneal,intranasal, transbuccal, transdermal, rectal, transalveolar(inhalation), or oral administration. Any suitable composition of theinvention, as described herein, may be used. In some embodiments, thecomposition is a neurotrophin covalently linked to a chimeric HIR-MAb.In some embodiments, the neurotrophin is a BDNF. In some embodiments,the BDNF is a variant as described herein, such as a carboxyl-terminaltruncated variant. In other embodiments, the neurotrophin is a GDNF(e.g., mature human GDNF).

A “disorder of the CNS” or “CNS disorder,” as those terms are usedherein, encompasses any condition that affects the brain and/or spinalcord and that leads to suboptimal function. In some embodiments, thedisorder is an acute disorder. Acute disorders of the CNS include focalbrain ischemia, global brain ischemia, brain trauma, spinal cord injury,acute infections, status epilepticus, migrane headache, acute psychosis,suicidal depression, and acute anxiety/phobia. In some embodiments, thedisorder is a chronic disorder. Chronic disorders of the CNS includechronic neurodegeneration, retinal degeneration, depression, chronicaffective disorders, lysosmal storage disorders, chronic infections ofthe brain, brain cancer, stroke rehabilitation, inborn errors ofmetabolism, autism, mental retardation. Chronic neurodegenerationincludes neurodegenerative diseases such as prion diseases, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), transversemyelitis, motor neuron disease, Pick's disease, tuberous sclerosis,lysosomal storage disorders, Canavan's disease, Rett's syndrome,spinocerebellar ataxias, Friedreich's ataxia, optic atrophy, and retinaldegeneration, and aging of the CNS.

In some embodiments, the invention provides methods of treatment of theretina, or for treatment or prevention of blindness. The retina, likethe brain, is protected from the blood by the blood-retinal barrier(BRB). The insulin receptor is expressed on both the BBB and the BRB,and the HIRMAb has been shown to deliver therapeutics to the retina viaRMT across the BRB. BDNF is neuroprotective in retinal degeneration, butit was necessary to inject the neurotrophin directly into the eyeball,because BDNF does not cross the BRB. In some embodiments, fusionproteins of the invention are used to treat retinal degeneration andblindness with a route of administration no more invasive than anintravenous or subcutaneous injection, because the HIRMAb delivers theBDNF across the BRB, so that the neurotrophin is exposed to retinalneural cells from the blood compartment.

In some embodiments, the invention provides a method of treatment fordepression. A subset of patients with depression may have a braindeficiency of BDNF, and the correlation of single nucleotidepolymorphisms (SNPs) with affective disorders has been reported. Thedirect injection of BDNF into the brain has durable anti-depressanteffects in rodent model. The BDNF must be injected directly into thebrain, because the neurotrophin does not cross the BBB. In someembodiments, the invention provides a method for treating depression bychronic administration of a fusion protein of the invention, thuselevating the brain levels of BDNF and being therapeutic in thosepatients with depression and a reduced production of brain BDNF.

Formulations and administration. Any suitable formulation, route ofadministration, and dose of the compositions of the invention may beused. Formulations, doses, and routes of administration are determinedby those of ordinary skill in the art with no more than routineexperimentation. Compositions of the invention, e.g., GDNF fusionproteins are typically administered in a single dose, e.g., anintravenous dose, of about 0.01-1000 mg, or about 0.05-500 mg, or about0.1-100 mg, or about 1-100 mg, or about 0.5-50 mg, or about 5-50 mg, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 25, 40, 45,50, 60, 70, 80, 90, or 100 mg. Typically, for the treatment of acutebrain disease, such as stroke, cardiac arrest, spinal cord injury, orbrain trauma, higher doses may be used, whereas for the treatment ofchronic conditions such as Alzheimer's disease, Parkinson's disease,Huntington's disease, MS, ALS, transverse myelitis, motor neurondisease, Pick's disease, tuberous sclerosis, addiction (e.g., drugaddiction), lysosomal storage disorders, Canavan's disease, Rett'ssyndrome, spinocerebellar ataxias, Friedreich's ataxia, optic atrophy,and retinal degeneration, and aging, lower, chronic dosing may be used.Oral administration can require a higher dosage than intravenous orsubcutaneous dosing, depending on the efficiency of absorption andpossible metabolism of the protein, as is known in the art, and may beadjusted from the foregoing based on routine experimentation.

For intravenous or subcutaneous administration, formulations of theinvention may be provided in liquid form, and formulated in saline basedaqueous solution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate.

Dosages for humans can be calculated from appropriate animal data. Forexample, human dosing of a BDNF-MAB conjugate is based on pre-clinicalpharmacokinetic studies, and these measurements have been performed in 2species, rats, and Rhesus monkeys. Prior work in 3 models of cerebralischemia in rats demonstrated the range of effective doses of theBDNF-MAb conjugate is 5-50 μg/rat or 20-200 μg/kg of BDNF in the form ofthe BDNF-MAb conjugate. Since the BDNF component of the fusion proteinmolecule is 16%, and the HIRMAb component is 84%, the dose of fusionprotein is 6-fold greater than the equivalent BDNF dose. Pharmacokineticstudies in rats show these doses produce a concentration of the BDNF inthe form of conjugate in plasma of 50-500 ng/mL, and in brain of 5-50ng/g. Pharmacokinetic studies in adult Rhesus monkeys with the HIRMAbshow that the average plasma concentration in the first hour is 0.1%injected dose (ID)/mL, and that the brain concentration is 0.02% ID/g.The brain concentration of the fusion protein is about 0.01% ID/g (FIG.27 ). Owing to the scaling effect between species, and to the 10-foldlarger body size and brain size of humans relative to Rhesus monkeys,the projected plasma and brain concentrations in humans are 0.01% ID/mLand 0.001% ID/g respectively. Since the human brain is 1200 grams,then >1% of the injected dose is delivered to the human brain, which isa level of brain uptake comparable to small molecules. Given an injecteddose of fusion protein of 2.5-25 mg in humans, the expected 60 minplasma concentration is 250-2500 ng/ml of fusion protein, and theexpected 60 min brain concentration is 25-250 ng/g of fusion protein,which is equivalent to 4-40 ng/gram brain of BDNF. The 5 mg and 25 mgfusion protein doses in humans will produce a brain concentration of theBDNF that is neuroprotective in either global or regional brainischemia. Since the BDNF comprises 16% of the fusion protein, theeffective doses of BDNF administered to humans is 0.4 or 4.0 mg,respectively, for the 2.5 or 25 mg dose of fusion protein.

The fusion protein may also be formulated for chronic use for thetreatment of a chronic CNS disorder, e.g., neurodegenerative disease,stroke or brain/spinal cord injury rehabilitation, or depression.Chronic treatment may involve daily, weekly, bi-weekly administration ofthe composition of the invention, e.g., fusion protein eitherintravenously, intra-muscularly, or subcutaneous in formulations similarto that used for acute treatment. Alternatively, the composition, e.g.,fusion protein may be formulated as part of a bio-degradable polymer,and administered on a monthly schedule.

Combination therapies. The composition of the invention, e.g., fusionprotein may be administered as part of a combination therapy. Thecombination therapy involves the administration of a composition of theinvention in combination with another therapy for the CNS disorder beingtreated. If the composition of the invention is used in combination withanother CNS disorder method or composition, any combination of thecomposition of the invention and the additional method or compositionmay be used. Thus, for example, if use of a composition of the inventionis in combination with another CNS disorder treatment agent, the two maybe administered simultaneously, consecutively, in overlapping durations,in similar, the same, or different frequencies, etc. In some cases acomposition will be used that contains a composition of the invention incombination with one or more other CNS disorder treatment agents.

Other CNS disorder treatment agents that may be used in methods of theinvention include, without limitation, thromolytic therapy for stroke,amyloid-directed therapy for Alzheimers disease, dopamine restorationtherapy for Parkinsons disease, RNA interference therapy for geneticdisorders, cancer, or infections, and anti-convulsant therapy forepilepsy. Dosages, routes of administration, administration regimes, andthe like for these agents are well-known in the art.

In some embodiments, the composition, e.g., fusion protein isco-administered to the patient with another medication, either withinthe same formulation or as a separate composition. For example, thefusion protein could be formulated with another fusion protein that isalso designed to deliver across the human blood-brain barrier arecombinant protein other than BDNF (e.g., GDNF). The fusion protein maybe formulated in combination with other large or small molecules.

VIII. Kits

Compositions of the invention, e.g., fusion proteins, may be provided asa kit that includes the formulation, e.g., fusion protein in a containerand in suitable packaging. The composition can be provided in a drypowder form, in solid form (i.e., lyophilized), in solution, or insuspension. If the composition is a protein, to the proteins may havebeen added emulsifiers, salts, preservatives, other proteins, nucleicacids, protease inhibitors, antibiotics, perfumes, polysaccharides,adhesive agents, polymers, microfibrils, oils, etc. The composition ispackaged for transport, storage and/or use by a consumer. Such packagingof therapeutic compositions for transport, storage, and use iswell-known in the art. Packaged compositions may include furthercomponents for the dispensing and storage of the composition, and mayalso include separately packaged diluent comprised of, e.g., sterilewater or a suitable buffer, for solubilizing the formulation, e.g.,fusion protein prior to administration to the patient. Kits of theinvention may also include written materials, including instructions foruse, results of clinical studies, desired outcome and expected course oftreatment, information about precautions and side effects, and the like.The kits may optionally further contain other components, such asgloves, scissors, tape, implements for disposal of used vials and otherwaste, masks, antiseptic, antibiotics, and the like.

EXAMPLES Example 1

Construction of the Single Tandem Vector Containing Complete Genes forIgG-Neurotherapeutic Fusion

Genetic engineering of a eukaryotic expression vector encoding the heavychain (HC) of the fusion protein is outlined in FIG. 1 . The finalfusion protein HC expression vector was designated pHIRMAb-BDNF, orclone 416. This vector was designed to produce a fusion protein,comprised of a BDNF variant fused to the HC of the HIRMAb. Either BDNFor a variant of BDNF (vBDNF) can be fused to the HIRMAb. The vBDNFdiffers from native human BDNF by substitution of certain amino acids,such as a vBDNF where the 2 amino acids at the carboxyl terminus of BDNFare absent in vBDNF. The clone 416 plasmid was derived from clone 400,which produces the HC of the chimeric form of the HIRMAb, and a cDNAencoding mature human vBDNF, which was produced as described in FIG. 2 .Clone 400 encodes a chimeric human IgG1 that is derived from achromosomal fragment encoding the human IgG1 constant region, and iscomprised of both intron and exon sequences. The HC gene of the chimericHIRMAb in clone 400 was subcloned at the BamHI site of the pCR IIplasmid to facilitate engineering of the stop codon located at the3′-end of the CH3 region by site directed mutagenesis (SDM). Theengineering of the stop codon located at the end of the CH3 region wasperformed by site-directed mutagenesis to produce a SspI site. The SspIsite allows for insertion of the vBDNF cDNA (FIG. 3 ) by blunt-endligation into clone 400 to form clone 415. SDM was performed using theQuickChange SDM kit (Stratagene, Calif.). Sense and complementarymutagenic primers were designed in a way that the CH3 stop codon(aaTGAg) is mutated to SspI site (aaTATt). In addition, primerscontained 15 nucleotides of the stop codon 5′- and 3′-surroundingregion; the sequence of these primers, designated SDM-SspI forward (FWD)and reverse (REV) are given in Table 1.

TABLE 1Nucleotide sequence of oligodeoxynucleotides used for engineeringplasmid clone 416 SDM-SspI-FWD (SEQ ID NO. 1)CCTGTCTCCGGGTAAATATTTGCGACGGCCGGCAAG SDM-SspI-REV (SEQ ID NO. 2)CTTGCCGGCCGTCGCAAATATTTACCCGGAGACAGG XhoI-NheI linker FWD (SEQ ID NO. 3)ATGCTCGAGGAATTCCCATGGATGATGGCTAGCAAGCTTATGXhoI-NheI linker REV (SEQ ID NO. 4)CATAAGCTTGCTAGCCATCATCCATGGGAATTCCTCGAGCAT XhoI-NheI (underlined) is aUniversal linker that contains the following RE sites:XhoI-EcoRI-NcoI-NheI-HindIII. SDM = site-directed mutagenesis; FWD= forward; REV = reverse

DNA sequence analysis of the IgG promoter region revealed the presenceof additional SspI sites in this region. Therefore, it was firstnecessary to release the HC promoter region (PRO-VH) by digestion ofclone 404 with XhoI and NheI, and the clone 404 was re-closed with aXhoI-NheI linker which produced clone 405 (-Pro-VH). The sequence of theforward and reverse ODNs used to produce the XhoI-NheI linker are givenin Table 1. Plasmid clone 405 (-Pro-VH) now carries the single SspI siteintroduced by SDM. The human vBDNF cDNA was subcloned at SspI to form anintermediate plasmid named clone 414 (not shown). The complete fusionprotein HC expression cassette was then reconstructed by subcloning ofthe PRO-VH fragment previously deleted to form clone 415. The fusionprotein HC gene was then subcloned in the eukaryotic expression vector,clone 400, at the BamHI site to form clone 416.

The vBDNF cDNA was produced by PCR via either of 2 equivalentapproaches. In one approach, a prokaryotic expression plasmid, pHTBS01,isolated as an expressed sequence tag (EST), and encoding human BDNF,was digested with BamHI and BpII, and gel purified, and re-ligated withT4 ligase and the 5′-end linker to produce clone 412 (FIG. 2 ). Thesequence of the forward and reverse ODNs used to produce the 5′-endlinker are given in Table 2.

TABLE 2 Engineering of 5’- and 3’-end linkers of vBDNF cDNA1) 5’-end linker of vBDNF FWD-ODN (SEQ ID NO. 5)TCCGGATCCTCGCGAGTATCACTCTGACCCTGCCCGTCGAGGTGAGCTGAGCGTG2) 5’-end linker of vBDNF REV-ODN (SEQ ID NO. 6)CACGCTCAGCTCACCTCGACGGGCAGGGTCAGAGTGCATACTCGCGAGGATCCGGA3) 3’-end linker of vBDNF FWD-ODN (SEQ ID NO. 7)AGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGTGATCGCGACTCGAGATG4) 3’-end linker of vBDNF REV-ODN (SEQ ID NO. 8)CATCTCGAGTCGCGATCACCTTTTAATGGTCAATGTACATACACAAGAAGTGTCTATCCTTATGAATCGCCAGCCAATTCTCTTTTTGCTATCCATGGTAAGGGCCCGCACGTACGACT5) VBDNF-PCR-U87 FWD-ODN (SEQ ID NO. 9) ATCTCGCGAGTATGCACTCTGACCCTGCC6) VBDNF-PCR-U87 REV-ODN (SEQ ID NO. 10) ATC TCGCGATCACCTTTTAATGGTCAASEQ ID NO 5 and 6: Artificial forward (FWD) and reverse (REV)oligodeoxynucleotide (ODN) duplex linkers were designed to engineer amature vBDNF cDNA that allows for insertion into the CH3 open readingframe (orf) of clone 400 heavy chain (HC) to form clone 416 (FIG. 1).The 5’-end linker is flanked by BamHI and EspI, respectively, and itreconstructs the amino terminus of the mature vBDNF. BamHI and EspIallow for directional subcloning into the vBDNF intermediate plasmidclone 413 (FIG. 2). A Nrul site follows BamHI and it enables insertionof the vBDNF into the HC vector (clone 405, FIG. 1) at the SspI site. Inaddition, the linker also has “GT” immediately after NruI to maintainthe orf of the CH3 (FIG. 1). This modification introduces a Ser-Ser-Metlinker between CH3 and the vBDNF amino terminus. SEQ ID NO 7 and 8: The3’-end linker contains SplI and XhoI to reconstruct the COOH terminus ofthe mature vBDNF and introduces a stop codon “TGA”. This linker hasSplI, XhoI and NruI sites for directional subcloning and insertion intoclone 405 (FIGS. 1 and 2). SEQ ID NO 9 and 10: FWD ODN reconstructs theamino terminus of the mature vBDNF and introduces a Ser-Ser-Met linker.NruI site for insertion into the expression vector is underlined. REVODN introduces the TGA stop codon. NruI site for insertion into theexpression vector is underlined.

Clone 412 was then digested with XhoI and BsiWI, and gel purified, andre-ligated with T4 ligase and the 3′end linker to produce clone 413(FIG. 2 ). The sequence of the forward and reverse ODNs used to producethe 3′-end linker are given in Table 2. The vBDNF cDNA, encoding thevBDNF with a reconstructed stop codon, was released from clone 413 byNruI, and gel purified; the ethidium bromide stain of the agarose gel isshown in FIG. 3A. This gel shows the expected size of the vBDNF cDNA,0.4 kb, and the vector backbone, 3.5 kb. Alternatively, the BDNF cDNAwas produced by PCR from cDNA derived by reverse transcription ofpolyA+RNA isolated from human U87 glioma cells, which produceneurotrophins. The primers used to produce the vBDNF by PCR from theU87-derived cDNA are given in Table 2. This PCR produced the expected0.4 kb vBDNF cDNA (FIG. 3B). The 0.4 kb vBDNF fragment was then digestedwith NruI, and subcloned into clone 415, as described in FIG. 1 , toproduce the full fusion protein HC expression cassette, which wasreleased by BamHI and subcloned into the original eukaryotic expressionplasmid to produce clone 416 (FIG. 1 ), the final expression plasmid forthe fusion protein HC. Clone 416 was analyzed by double digestion withNheI and BamHI and compared with that of the original clone 400, whichlacks the vBDNF. The agarose gel-separated products are shown in FIG. 3C, where lanes 1 and 3 show the fragments generated from clone 416 andclone 400, respectively. Both plasmids produce a 6 kb vector backbone(upper of 3 bands in lanes 1 and 3), and a 2.5 kb promoter region (lowerof 3 bands in lanes 1 and 3). However, the size of the middle band is0.4 kb larger for clone 416, as compared to clone 400 (middle band,lanes 1 and 3). A negative clone is shown in lane 2 of FIG. 3C.

The nucleotide and amino acid sequence of the reconstructed carboxylterminus at the CH3 region of the HIRMAb HC, a 3-amino acid linker(Ser-Ser-Met), the vBDNF sequence, followed by a stop codon is shown inFIG. 4 . The entire 2711 nucleotides (nt) comprising the fusion proteinHC gene of clone 416 is shown in FIG. 5 . The ATG initiation codon andthe TGA stop codon are underlined. The human IgG1 constant region intronand exon sequences are shown in italics and bold font, respectively, inFIG. 5 . The vBDNF nt sequence in the clone 416 vector is underlined inFIG. 5 . These data show that intronic sequence is found between CH1 andthe hinge region, between the hinge region and CH2, and between CH2 andCH3 regions of the human IgG1 constant region. The open reading frame(orf) of the fusion protein HC gene encodes for a 563 amino acidprotein, following cleavage of a 19 amino acid signal peptide, and theamino acid sequence of the fusion protein HC is shown in FIG. 6 . Thesignal peptide is underlined; the cysteine (C) residues within theconstant region that form inter- or intra-chain disulfide bridges areshown in bold font; the serine-serine-methionine (SSM) linker betweenthe CH3 region of the IgG and the vBDNF is underlined; the singleN-linked glycosylation site, at the asparagine residue within CH2 inshown by bold underlined font (FIG. 6 ). The amino acid sequences of theindividual domains of the fusion protein HC protein are given in FIG. 7. The vBDNF domain of the fusion protein is comprised of 117 aminoacids.

Clone 416 plasmid DNA was electroporated into mouse myeloma cells thathad previously been transfected with an expression plasmid encoding thelight chain (LC) of the chimeric HIRMAb. Since the vBDNF is fused onlyto the HC, there is no modification of the LC of the chimeric HIRMAb.Following selection of transfected cell lines, media from 96-well plateswere screened with an ELISA comprised of 2 anti-human IgG antibodies;one antibody is directed against the heavy chain of human IgG1, and theother antibody is directed against human kappa light chains Myelomaclones encoding for intact fusion protein were isolated, and propagatedin a 10 L bioreactor. However, the production levels of the fusionprotein were low. This low production was attributed to several factors,including (i) transfection of the myeloma line by 3 separate expressionplasmids encoding the heavy chain gene, the light chain gene, and theantibiotic resistance gene; and (ii) the use of genomic fragment of theheavy and light chain genes with large intronic sequences. Therefore,the fusion protein expression plasmid was re-engineered with thefollowing features:

-   (1) the polymerase chain reaction (PCR) was used to convert genomic    fragments of the fusion protein HC and LC genes into ‘intron-less’    cDNA forms of the 2 genes-   (2) the cDNA forms the fusion protein HC and LC genes were placed on    a single ‘tandem vector’ in which the 2 genes were placed in    separate and tandem expression cassettes with separate promoters-   (3) the promoter driving the expression of the fusion protein HC and    LC genes was changed from the human IgG promoters to the    cytomegalovirus (CMV) promoter, to enable transfection of    non-myeloma cells, such as Chinese hamster ovary (CHO) cells-   (4) the tandem vector encoding fusion protein contains a gene    encoding for the dihydrofolate reductase (DHFR) gene, under a    separate SV40 promoter, to allow for methotrexate (MTX) selection of    CHO lines which contain amplification of the genome in the region of    the insertion of the expression vector.

In order to produce the fusion protein tandem vector, it was firstnecessary to produce intermediate plasmids, which separately encode cDNAforms of the fusion protein HC and LC genes. Eukaryotic expressionplasmids carrying the CMV promoter and the bovine growth hormone (BGH)poly-A (pA) transcription termination sequences, and designated pCD,were digested with NheI and XhoI and re-ligated with T4 ligase and anNheI-EcoRV-KpnI-ScaI-BamHI-XhoI linker, as shown in FIG. 8 . Thesequence of the forward and reverse ODNs used to produce this linker aregiven in Table 3.

TABLE 3 Nucleotide sequence of ODNs used for engineering of intronlessexpression vectors1) Linker NheI-EcoRV-KpnI-XcaI-BamHI-XhoI FWD ODN (SEO ID NO. 11)ATGGCTAGCGATATCGGTACCGTATACGGATCCCTCGAGATG2) Linker NheI-EcoRV-KpnI-XcaI-BamHI-XhoI REV ODN (SEO ID NO. 12)CATCTCGAGGGATCCGTATACGGTACCGATATCGCTAGCCAT3) PCR cloning of LC FWD ODN primer (SEO ID NO. 13)GTGACAAACACAGACATAGGATATC4) PCR cloning of LC REV ODN primer (SEQ ID NO. 14)ATGCTCGAGCTAACACTCTCCCCT5) PCR cloning of fusion protein HC FWD ODN primer (SEQ ID NO. 15)ATGAATATTCCACCATGGAATGCAGC6) PCR cloning of fusion protein HC REV ODN primer (SEQ ID NO. 16)ATAGGATCCTCACCTTTTAATGGTCAA RE cloning sites are underlined: GATATC:EcoRV, CTCGAG: XhoI, AATATT: SspO, GGATCC: BamHI.

The resulting plasmid, designated pCD-linker (FIG. 8 ) was digested withEcoRV and BamHI and reclosed with T4 ligase and the fusion protein HCcDNA generated by PCR. For the PCR reaction, the above mentioned myelomaline that had been dual transfected with genomic constructs of thefusion protein HC (clone 416) and LC genes were digested and myelomaderived polyA+ RNA was produced (part A in FIG. 8 ). Oligodeoxythymidine(ODT) primers were used to produced myeloma cDNA with reversetranscriptase from 0.5 ug of myeloma polyA+RNA, followed by a finalRNase digestion. From this cDNA, PCR was used to produce the cDNA formof the fusion protein HC gene, using the forward and reverse primersshown in Table 3, and high fidelity Pfu DNA polymerase. Similarly, thefusion protein LC cDNA was produced by PCR from the myeloma derivedcDNA, and the sequences of the forward and reverse PCR primers used toamplify the fusion protein LC cDNA are given in Table 3. Following PCR,the cDNA was applied to an 0.8% agarose gel, and all amplificationsyielded a single product, a 1.8 kb fusion protein HC cDNA (lane 1, FIG.3D), and a 0.7 kb fusion protein LC cDNA (lane 2, FIG. 3D). The fusionprotein HC PCR product was digested with SspI and BamHI and subclonedinto CD-linker to produce the clone 422a (FIG. 8 ), which is anintronless eukaryotic expression plasmid encoding the fusion protein HCcDNA. Clone 422a was analyzed by restriction endonuclease using NheI;digestion with this enzyme, which has a site in the new multiple cloningregion of the pCD vector, produced the expected 0.4 kb fragmentcorresponding to the fusion protein heavy chain variable region (VH)cDNA (lanes 1-4, FIG. 3E). The nucleotide sequence of the fusion proteinHC cDNA encoded by clone 422a is shown in FIG. 9A, which shows theintron sequences present in clone 416 (FIG. 5 ) have been deleted by thePCR of processed myeloma RNA. The amino acid sequence encoded by thefusion protein HC cDNA is given in FIG. 9B, and this amino acid sequenceis identical to that produced by the genomic fragment in clone 416 (FIG.6 ).

The fusion protein LC PCR product was digested with EcoRV and XhoI andsubcloned into CD-linker to produce the clone 423a (FIG. 10 ), which isan intronless eukaryotic expression plasmid encoding the fusion proteinLC cDNA. Clone 423a was analyzed by restriction endonuclease using EcoRVand BamHI; digestion with these enzymes, which have a site in the newmultiple cloning region of the pCD vector, produced the expected 0.7 kbfragment corresponding to the fusion protein LC cDNA (lanes 1-5, FIG.3F). The nucleotide sequence of the fusion protein LC cDNA encoded byclone 423a is shown in FIG. 11A, which shows the intron sequences havebeen deleted by the PCR of processed myeloma RNA. The amino acidsequence encoded by the fusion protein LC cDNA is shown in FIG. 11B.

Clones 422a and 423a were the precursors to the fusion protein tandemvector, as outlined in FIG. 12 . In 2 steps, clone 422a was subjected toSDM to introduce an EcoRI site at the 3′-end of the fusion protein HCexpression cassette; the sequences of the forward and reverse SDMprimers are given in Table 4.

TABLE 4 Nucleotide sequences of ODNs used for engineering of TV-121) EcoRI-SDM FWD ODN (SEP ID NO. 17)AAAAGGCCAGGAACCGAATTCAGATCTCGTTGCTGGCGTTTT2) EcoRI-SDM REV ODN (SEP ID NO. 18)AAAACGCCAGCAACGAGATCTGAATTCGGTTCCTGGCCTTTT3) EcoRI linker FWD (SEP ID NO. 19)ATCGAATTCAAGCTTGCGGCCGCGTATACAGATCTATC4) EcoRI linker REV (SEP ID NO. 20)GATAGATCTGTATACGCGGCCGCAAGCTTGAATTCGAT EcoRI site in EcoRI-SDM PDN isunderlined. The EcoRI linker introduces EcoRI-HindIII-NotI-XcaI REsites.

In step 2, the mutated clone 422a was digested with EcoRI, blunt-ended,and re-ligated with the EcoRI-HindIII-NotI-XcaI linker to produce clone422a-I (FIG. 12 ). The sequence of the ODNs used to produce this EcoRIlinker are given in Table 4. Clone 422a-I was digested with EcoRI andHindIII, and closed with T4 ligase in the presence of the fusion proteinLC expression cassette to produce clone 422a-II (FIG. 12 ). The fusionprotein LC expression cassette was generated by digestion of clonepBS-LC-1 with EcoRI and HindIII Clone pBS-LC-1 was produced fromEcoRV-digested pBS (Bluescript), T4 ligase, and the fusion protein LCexpression cassette produced by digestion of clone 423a with SspI (FIG.12 ). In parallel, a mouse DHFR expression cassette, containing the SV40promoter and the hepatitis C virus polyA region, was produced from thepFR400 plasmid (designated pDHFR) by digestion of the plasmid with SmaIand SalI (FIG. 12 ). The final fusion protein tandem vector was producedby subcloning the DHFR expression cassette into XcaI digested clone422a-II followed by closure with T4 ligase (FIG. 12 ). The fusionprotein tandem vector was analyzed by restriction endonuclease, and the11 kb plasmid was linearized by PvuI (lane 1, FIG. 3G). The 1.8 kbfusion protein LC and 1.5 kb DHFR expression cassettes, and the 8 kbvector backbone including the fusion protein HC expression cassette werereleased by digestion with EcoRI and HindIII (lane 2, FIG. 3G). Thetandem vector was subjected to DNA sequencing in both directions, andthe nucleotide sequence, and the deduced amino acid sequence of thefusion protein HC, the fusion protein LC, and the DHFR genes are shownin FIGS. 14, 15 , and 16, respectively. The calculated MW of the fusionprotein HC and LC are 62,220 and 25,760 Da, respectively, not accountingfor any carbohydrate content of the fusion protein HC.

Example 2

Electroporation of CHO cells with fusion protein tandem vector andcultivation in a bioreactor. The fusion protein tandem vector (FIG. 12 )was linearized with PvuI and electroporated into CHO-K 1 cells followedby selection with G418 (375 ug/ml) for 3 weeks. Positive clones weredetected in 96 well plates with a human IgG ELISA that uses 2 primaryantibodies to both the human IgG1 HC and the human kappa LC. Cell linesof high copy number of the transgene were selected by graded increasesin MTX to 600 nM. The MTX-selected cell line was grown in T175 flasksand then transferred to a 20 L bioreactor with a 10 L volume of CHO cellserum free medium (SFM). As shown in FIG. 17 , the CHO cells weremaintained at high density in excess of 10 million viable cells/mL fornearly 50 days in perfusion mode in the bioreactor. The secretion bythese cells of the fusion protein was detected by ELISA using antibodiesto either human IgG or to human BDNF. As shown in FIG. 18 , the fusionprotein is a 1:1 fusion of the vBDNF to the carboxyl terminus of theHIRMAb heavy chain, which results in formation of the fusion proteinheavy chain This heavy chain complexes with the light chain, as shown inFIG. 18 . Therefore, the fusion protein should react equally well to 3antibodies directed against: (i) the human IgG1 HC, (ii) the human kappaLC; or (iii) human BDNF. As shown in FIG. 19 , there is a directcorrelation in measurement of the fusion protein in the CHO cell mediumdepending on whether anti-human IgG or anti-human BDNF antibodies areused in the ELISA. These ELISA results were confirmed withimmunocytochemistry (ICC), which showed the CHO cells transfected withTV-120 were immunoreactive with antibodies to either human IgG or tohuman BDNF, and that the BDNF immune signal was eliminated by absorptionof the anti-BDNF antibody with recombinant BDNF.

Example 3

Purification and characterization of bioreactor produced fusion protein.The conditioned medium obtained from the bioreactor under perfusion modewas passed through a 1 μm filter, and the medium collected in a 200 LBioprocess container under sterile conditions, which were maintained at4° C. in a glass door refrigerator contiguous with the bioreactor. Then,200 L batches of conditioned medium were passed through 1 μm and 0.4 μmpre-filters for the removal of cell debris. The medium was thenconcentrated with tangential flow filtration (TFF). The TFF system was aPellicon 2 model from Millipore and was comprised of five 0.5 m²filtration cassettes with a 30 kDa molecular weight cutoff and a totalsurface area of 2.5 m². A transmembrane gradient of 15 PSI was produced,which results in a reduction in volume of the 200 L to 2 L within 2hours. The concentrated medium was passed through an 0.22μ filter priorto elution through 100 mL Prosep A (Millipore) recombinant protein Aaffinity column. Following application of the sample, the column waswashed with buffer A (0.025 M NaCl, 0.025 M Tris, pH=7.4, 3 mM EDTA).The elution of CHO cell host protein (CHOP) was monitored at A280 with aShimadzu detector. The fusion protein was eluted with 0.1 M citric acid(pH=3) in tubes containing Tris base to cause immediate neutralizationto pH 7. The neutralized acid eluate pool was diluted with doubledistilled water until the conductivity was <7 mS, and the material wasapplied to a 50 mL Sepharose SP cation exchange column (Amersham) thathas been equilibrated with a 0.02 M Tris, pH=7.5. Following washing inthe Tris buffer, the residual CHOP was separated from the fusion proteinwith a linear NaCl gradient from 0 to 1 M NaCl. The fusion protein peakwas pooled and buffer exchanged and concentrated with a Milliporediafiltration unit with a 30 kDa molecular weight cutoff. The finalconcentrated antibody solution was sterile filtered (0.22 μm) and storedat 4° C. The fusion protein was purified to homogeneity on sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), asdemonstrated in FIG. 20 . The size of the fusion protein heavy chain was68 kDa as compared to the size of the HIRMAb heavy chain, which was 54kDa. The difference between the size of the fusion protein and HIRMAbheavy chains reflects the added vBDNF monomer (14 kDa) fused to eachheavy chain of the fusion protein. The fusion protein reacts with bothanti-human IgG antibodies and anti-human BDNF antibodies on Westernblotting with the expected molecular weight size of the immunoreactivebands (FIG. 21 ). Isoelectric focusing (IEF) shows the isoelectric point(pI) of recombinant BDNF was highly cationic with a pI>10 (FIG. 22 ).The observed pI of the fusion protein was 8.5, and approximates the pIof the HIRMAb (FIG. 22 ). The observed pI of the fusion protein, 8.5,was consistent with the calculated pI, which is 9.04 and 5.27 for thefusion protein HC and LC, respectively (http://scansite.mit.edu/).

Example 4

The fusion protein is bi-functional and binds with high affinity to boththe human insulin receptor and to the human trkB receptor. The affinityof the fusion protein for the HIR extracellular domain (ECD) wasdetermined with a competitive ligand binding assay (CLBA) using thelectin affinity purified HIR ECD. CHO cells transfected with the HIR ECDwere grown in serum free media (SFM), and the HIR ECD was purified witha wheat germ agglutinin affinity column. The HIR ECD was plated onNunc-Maxisorb 96 well dishes and the binding of the murine HIRMAb to theHIR ECD was detected by radioactivity measurements following addition of[¹²⁵I] murine HIRMAb as the ligand in the binding assay (FIG. 23A). Thebinding of the [¹²⁵I] murine HIRMAb to the HIR ECD was displaced by theaddition of unlabeled fusion protein or HIRMAb as demonstrated in FIG.23B. The CLBA shows comparable binding of the HIRMAb or the fusionprotein. A Scatchard analysis using a high affinity and low affinitybinding site model and nonlinear regression analysis was performed todetermine the affinity constant of the fusion protein binding to theHIR. Both the fusion protein and the HIRMAb bind equally well to the HIRwith a high affinity binding constant, Ki=0.63±0.07 nM (FIG. 23B).

The TrkB CLBA was designed for measurement of the affinity of the fusionprotein for recombinant human TrkB ECD. The design of a TrkB CLBA wasmade difficult by the cationic nature of BDNF, which causes a highdegree of nonspecific binding in the assay and this reduces thesensitivity of the assay. The nonspecific binding of BDNF could beeliminated by conjugation of 2000 Da polyethyleneglycol (PEG) to theprotein. A bifunctional PEG molecule, biotin-PEG²⁰⁰⁰-hydrazide (Hz), wascommercially obtained, and conjugated to BDNF to produceBDNF-PEG²⁰⁰⁰-biotin, as outlined in FIG. 24A; this molecule was used asthe “tracer” in the CLBA. The TrkB ECD was absorbed to ELISA plates andbinding of BDNF-PEG²⁰⁰⁰-biotin to the TrkB was detected colorimetricallywith avidin and biotin peroxidase (FIG. 24A). Prior studies showed theELISA signal (A490) was directly proportional to the amount of TrkBadded to the well. In addition, the assay had a very low blank and theA490 was <0.04 when no TrkB is plated. The binding of theBDNF-PEG²⁰⁰⁰-biotin to the TrkB was competitively displaced by therecombinant BDNF (FIG. 24B) or the fusion protein (FIG. 24C). TheScatchard analysis of the binding data using nonlinear regressionanalysis allowed for the computation of the Ki of binding of either BDNFor fusion protein to TrkB, as shown in FIGS. 24B and 24C, respectively.The affinity of the fusion protein for TrkB was not statisticallydifferent from the affinity of the recombinant BDNF (FIG. 19 B,C). Thenonspecific binding (NSB) of the assay was comparable for either BDNF orthe fusion protein. The NSB likely represents nonlinear cooperativebinding of the neurotrophin to the TrkB extracellular domain The TrkBCLBA results shown in FIG. 24 indicate the affinity of fusion proteinfor the TrkB receptor was not changed following fusion of the vBDNF tothe carboxyl terminus of the HIRMAb heavy chain.

Neurotrophins such as BDNF require an obligatory formation of ahomo-dimeric structure to be biologically active, and to bind with highaffinity to the cognate receptor, e.g. TrkB. A naturally occurringhomo-dimeric structure between two BDNF molecules was formed when theneurotrophin was fused to a carboxyl terminus of the CH3 region of anIgG molecule, as illustrated in FIG. 18 . The surprising observation ofthe maintenance of the high affinity binding of BDNF for TrkB (FIG. 24), despite fusion to the HIRMAb heavy chain (FIG. 18 ), is consistentwith the fact that BDNF normally binds to TrkB as a dimer.

Example 5

Human neural cells subjected to hypoxia are neuroprotected by the fusionprotein with equal activity as recombinant BDNF. Human SH-SY5Y neuralcells were exposed to 10 uM retinoic acid for 7 days, which induces geneexpression of trkB, the BDNF receptor. The cells were then exposed to 16hours of oxygen deprivation in a sealed chamber, with oxygen sensor.Excitotoxic neural damage was then induced by 4 hours of re-oxygenation(FIG. 25A). During this 4 hour re-oxygenation period, the cells wereexposed to either no treatment or equi-molar concentrations of humanrecombinant BDNF or fusion protein. As shown in FIG. 25B, the fusionprotein was equipotent with native human BDNF with respect to inducingneuroprotection in human neural cells exposed to excitoxicischemia-re-oxygenation.

Example 6

High affinity binding of fusion protein to human blood-brain barrierinsulin receptor in isolated human brain capillaries. Isolated humanbrain capillaries are used as an in vitro model system of the human BBB(FIG. 26A). The fusion protein was radiolabeled with 3H-N-succinimidylpropionate, and added to the human brain capillaries to establish aradio-receptor assay (RRA) of fusion protein binding to the HIR of thehuman BBB. [³H]-fusion protein is specifically bound to the BBB, as thebinding is self-inhibited by unlabeled fusion protein (FIG. 26B). Thefusion protein is bound by the insulin receptor of the human BBB,because the murine HIRMAb (mHIRMAb) also inhibits binding of [³H]-fusionprotein to the human BBB. The binding data in FIG. 26B were fit to aScatchard plot with a non-linear regression analysis to produce thebinding constants: K_(D)=0.55±0.07 nM, B_(max)=1.35±0.10 pmol/mg_(p),and NSB=0.39±0.02 pmol/mg_(p), where K_(D) is the dissociation constant,Bmax is the maximal binding, and NSB is the non-saturable binding. TheKD is <1 nM, which indicate the fusion protein binds the HIR on thehuman BBB with very high affinity.

Example 7

Pharmacokinetics and brain uptake of fusion protein by the adult Rhesusmonkey. The fusion protein was tritiated with [³H]-N-succinimidylpropionate to a specific activity of 2.0 μCi/μg. A 5 year old femaleRhesus monkey, weighing 5.2 kg, was administered by a single intravenousinjection a dose of 746 μCi (373 μg), and serum was collected atmultiple time points over a 180 min period. The serum glucose of theanesthetized, overnight-fasted primate was constant throughout the 180min study period, and averaged 72±2 mg %, which indicates that theadministration of the HIRMAb based fusion protein caused no interferenceof the endogenous insulin receptor, and had no effect on glycemiacontrol.

The serum removed from the anesthetized Rhesus monkey was analyzed fortotal radioactivity (FIG. 27A), and radioactivity that was precipitableby trichloroacetic acid (TCA) (FIG. 27B). At 180 minutes after druginjection, the animal was euthanized, and brain radioactivity wasanalyzed with the capillary depletion method (FIG. 27C), similar toprior work on the brain uptake of [¹²⁵I]-labeled murine HIRMAb in theRhesus monkey. Based on the specific activity of the [³H]-fusionprotein, the brain radioactivity was converted to ng per gram (g) brain,as shown in FIG. 27D, and this level was compared to the reportedendogenous concentration of BDNF in the adult primate brain.

The plasma pharmacokinetics analysis (FIG. 27A) shows that the fusionprotein of the genetically engineered

HIRMAb and the BDNF is removed from blood at the same rate as theoriginal murine HIRMAb. This is an important finding, because it showsthat the fusion of BDNF, a highly cationic protein, to the HIRMAb doesnot accelerate the blood clearance of the HIRMAb. Prior work shows thatthe attachment of the cationic BDNF to a monoclonal antibody greatlyaccelerates the blood clearance of the antibody, owing to the cationicnature of the BDNF, which greatly enhances hepatic uptake. The work inFIG. 27A shows that when the cationic BDNF was re-engineered as an IgGfusion protein, the plasma pharmacokinetics was dominated by the IgGmoiety, and that the blood level of the BDNF remains high for aprolonged period.

The data in FIG. 27B show that when BDNF was re-formulated as an IgGfusion protein, the metabolic stability of the neurotrophin in blood wasgreatly enhanced, as compared to the native BDNF. Owing to its cationicnature, the native BDNF was rapidly removed from blood, and was rapidlydegraded into TCA-soluble radioactive metabolites (FIG. 27B). However,the TCA-insoluble form of the labeled fusion protein remains high duringthe 3 hours after an intravenous injection in the primate (FIG. 27B).The data in FIGS. 27A,B show the advantages of re-engineering aneurotrophin pharmaceutical as a fusion protein. The native neurotrophinwas rapidly removed from blood and was rapidly degraded. However, theplasma pharmacokinetics profile, and metabolic stability profile, of theneurotrophin resemble those of an IgG molecule, when theIgG-neurotrophin fusion protein was produced.

Native BDNF is not transported across the BBB. Similarly, a [³H]-mouseIgG2a isotype control antibody was not transported across the BBB in theadult Rhesus monkey, as the brain volume of distribution (V_(D)) of theIgG at 180 minutes after an intravenous injection was equal to theplasma volume, 18 μL/g (FIG. 27C, open bars). Conversely, the brainV_(D) of the [³H]-fusion protein exceeds 140 μl/g brain (FIG. 27C,closed bars). Capillary depletion analysis separates the brainvasculature from the post-vascular supernatant, and allows detection ofthe transport of a drug through the BBB and into brain, as opposed tosimple sequestration of the drug by the brain vasculature. The brainV_(D) of the post-vascular supernatant of the [³H]-fusion protein wasequal to the V_(D) of the brain homogenate (FIG. 27C), which indicatesthe fusion protein was transported through the BBB and into brainparenchyma.

The brain V_(D) of the fusion protein was converted into ng fusionprotein per gram brain, based on the specific activity of the[³H]-fusion protein, and this allowed for calculation of the total massof fusion protein in the brain, 24±1 ng/g, as shown in FIG. 27D. Thisvalue is >10-fold higher than the endogenous brain concentration of BDNFin the adult primate (45). Therefore, the administration of a dose of373 μg to a 5.2 kg Rhesus monkey, which is equal to a normalized dose of72 μg/kg of fusion protein, results in a marked increase in the brainconcentration of BDNF. Such an increase in brain BDNF, followingintravenous administration, is not possible with native BDNF, becausethe native BDNF does not cross the BBB. However, when BDNF isre-engineered in the form of the fusion protein, then pharmacologicallyactive levels of the neurotrophin in brain are achieved (FIG. 27D).

The data shows that: (1) the plasma mean residence time (MRT) of thefusion protein, 312 minutes, was 100-fold greater than the MRT fornative BDNF, which was 3.0 minutes, and (2) the systemic clearance ofthe fusion protein, 0.94 mL/min/kg, was 39-fold slower than the systemicclearance of the BDNF, which was 37 mL/min/kg. In other words, theaverage blood level of the recombinant protein was up to 100-foldgreater when the recombinant protein was re-formulated as an IgG fusionprotein. Thus, fusion of the BDNF to the molecular Trojan horse had 2benefits: (1) the molecular Trojan horse carried the BDNF across theblood-brain barrier (BBB), whereas the BDNF alone cannot cross the BBB,and (2) the molecular Trojan horse prevented the rapid loss from bloodof the neurotrophin; BDNF by itself lasts only about 3 minutes in theblood. Both of these properties serve to enhance the pharmacologicaleffect of the BDNF in brain following administration into the bloodstream. See, e.g., Table 5.

TABLE 5 Pharmacokinetic parameters for [³H]-fusion protein and [³H]-BDNFParameter [³H]-fusion protein [³H]-BDNF A₁ (% ID/ml) 0.147 ± 0.020  5.28± 0.60 A₂ (% ID/ml) 0.061 ± 0.005  2.26 ± 0.32 k₁ (min⁻¹) 0.195 ± 0.050 1.75 ± 0.26 k₂ (hr⁻⁴) 0.186 ± 0.042  15.6 ± 0.6 t_(1/2) ¹ (min)  3.5 ±0.9  0.42 ± 0.07 t_(1/2) ² (hr)  3.7 ± 0.9 0.045 ± 0.001 CL_(ss)(ml/min/kg)  0.94 ± 0.16  37.0 ± 2.5 MRT (min)   312 ± 78  3.0 ± 0.3 A₁,A₂, k₁, and k₂ are the intercepts and slopes of the bi-exponentialfunction describing the decay in plasma concentration with time. Theparameters for the fusion protein were determined for the Rhesus monkey,and the parameters for BDNF were determined in the adult rat. All dataare normalized for differences in body weight. t_(1/2) ¹ and t_(1/2) ²are computed from k₁ and k₂, respectively, and are the half-times of thedecay curves for each exponent. CL_(ss) and MRT are the steady stateclearance and mean residence time, respectively, and are computed fromA₁, A₂, k₁, and k₂ using standard pharmacokinetic formulations.

Example 8

Neuroprotection in regional brain ischemia by conjugates of BDNF and aBBB molecular Trojan horse. Numerous attempts have been made to developneuroprotective agents for the treatment of acute stroke. There havebeen no successes to date because the neuroprotective drugs are eithertoo toxic, in the case of certain small molecules, or ineffective,because the drug does not cross the BBB. BDNF is neuroprotective wheninjected directly in the brain in parallel with experimental stroke inrodents and regional brain ischemia. The BDNF must be injected acrossthe skull bone into the brain, because this large molecule drug does notcross the BBB. Since the BBB is intact in the early hours after regionalbrain ischemia, and since BDNF does not cross the BBB, then there is noneuroprotective effect in the ischemic brain following the intravenousadministration of BDNF alone. To deliver BDNF across the BBB, theneurotrophin was attached to a mouse MAb to the rat transferrin receptor(TfR). This peptidomimetic MAb carries BDNF across the BBB, and thecombined BDNF-MAb conjugate is highly neuroprotective following delayedintravenous administration in experimental stroke, because the BDNF isable to cross the BBB and enter the brain from blood. Once inside thebrain, and behind the BBB, the BDNF activates its cognate receptor,trkB, which then induces neuroprotection in ischemic neurons, and stopsthe apoptotic death cycle. The neuroprotective effect of the BDNF-MAbconjugate demonstrates a dose response effect, a time response effect,and is long-lasting, as the neuroprotection at 7 days is identical tothe neuroprotection at 1 day after a single intravenous administrationof the BDNF-MAb conjugate. See, e.g., Zhang and Pardridge (2001) BrainRes. 889:49-56, and Zhang and Pardridge (2001) Stroke 32:1378-1374,which are incorporated by reference herein in their entirety. The fusionprotein would also be neuroprotective in human stroke, since the BDNF isfused to an MAb to the HIR, which rapidly binds to both the human BBB invitro, and is rapidly transported across the primate BBB in vivo.

Example 9

Neuroprotection in global brain ischemia of conjugates of BDNF and a BBBmolecular Trojan horse. The direct injection of BDNF into the brain isalso neuroprotective in transient forebrain ischemia (TFI), such asmight occur after a cardiac arrest. However, intravenous BDNF is notneuroprotective in TFI, because the BDNF does not cross the BBB, andbecause the BBB is intact in the early hours after TFI, whenneuroprotection is still possible. Conversely, intravenous BDNF wasneuroprotective in TFI if the BDNF was attached to a mouse MAb againstthe rat transferrin receptor (TfR), which acts as a molecular Trojanhorse to ferry the BDNF across the BBB and into brain. Adult rats weresubjected to TFI, which resulted in a flat-line electroencephalogram(EEG) for approximately a 10-minute period. The animals wereresuscitated and then administered 1 of 4 different therapeuticsintravenously: (a) buffer, (b) unconjugated BDNF, (c) the receptorspecific MAb without the BDNF attached, and (d) the BDNF-MAb conjugate.In the case of the animals treated with saline, unconjugated BDNF, orMAb alone, there was no neuroprotection of pyramidal neurons in the CA1sector of hippocampus. However, in the case of the BDNF-MAb conjugate,there is complete normalization of CA1 pyramidal neuron densityfollowing delayed intravenous administration. See, e.g., Wu andPardridge (199), PNAS (USA) 96:254-259, which is incorporated byreference herein in its entirety. This shows that BDNF is stronglyneuroprotective in global brain ischemia following delayed intravenousadministration, providing the BDNF is attached to a BBB molecular Trojanhorse. The recombinant fusion protein of BDNF and a receptor specificMAb could be given following cardiac arrest to prevent permanent braindamage.

Example 10

BDNF is neuroprotective in brain and spinal cord injury if theneurotrophin can access brain cells. BDNF is neuroprotective in braininjury, providing the neurotrophin is injected directly through theskull bone, because BDNF does not cross the BBB. BDNF is alsoneuroprotective in brain subjected to excitotoxic injury by neurotoxins,and is neuroprotective in brain infected with the human immunedeficiency virus (HIV)-1. BDNF is also neuroprotective in acute spinalcord injury; however, the BDNF must be administered by direct infusioninto the spinal canal, because the BDNF does not cross the blood-spinalcord barrier, which is the same as the BBB in the forebrain. In allthese cases, the intravenous administration of BDNF would not beneuroprotective, because the BDNF does not cross the BBB, and the BBB isintact in brain injury in the early hours after the injury, whenneuroprotection is still possible. Conversely, the BDNF fusion proteinwould be neuroprotective in these conditions following intravenousadministration, because the BDNF is fused to the BBB molecular Trojanhorse, and is able to penetrate the brain and spinal cord from the bloodfollowing peripheral administration.

Example 11

BDNF is neuroprotective in chronic neurodegenerative conditions of brainif the neurotrophin can access brain cells. Neurotrophins, such as BDNFcan be developed as drugs for peripheral routes of administration,providing the neurotrophin is enabled to cross the BBB. Fusion of BDNFto the chimeric HIRMAb offers a new approach to the non-invasivedelivery of BDNF to the brain in humans for the chronic treatment ofneurodegenerative disease, including prion diseases, Alzheimer's disease(AD), Parkinson's disease (PD), Huntington's disease (HD), ALS,transverse myelitis, motor neuron disease, Pick's disease, tuberoussclerosis, lysosomal storage disorders, Canavan's disease, Rett'ssyndrome, spinocerebellar ataxias, Friedreich's ataxia, optic atrophy,and retinal degeneration, and brain aging.

Example 12

BDNF as a therapeutic in retinal degeneration and blindness. The retina,like the brain, is protected from the blood by the blood-retinal barrier(BRB). The insulin receptor is expressed on both the BBB and the BRB,and the HIRMAb has been shown to deliver therapeutics to the retina viaRMT across the BRB (Zhang et al, (2003) Mol. Ther. 7: 11-18). BDNF isneuroprotective in retinal degeneration, but it was necessary to injectthe neurotrophin directly into the eyeball, because BDNF does not crossthe BRB. The fusion protein could be used to treat retinal degenerationand blindness with a route of administration no more invasive than anintravenous or subcutaneous injection, because the HIRMAb would deliverthe BDNF across the BRB, so that the neurotrophin would be exposed toretinal neural cells from the blood compartment.

Example 13

BDNF as a therapeutic for depression. A subset of patients withdepression may have a brain deficiency of BDNF, and the correlation ofsingle nucleotide polymorphisms (SNPs) with affective disorders has beenreported. The direct injection of BDNF into the brain has durableanti-depressant effects in rodent model. The BDNF must be injecteddirectly into the brain, because the neurotrophin does not cross theBBB. The chronic administration of the fusion protein would provide ameans for elevating the brain levels of BDNF, and may be therapeutic inthose patients with depression and a reduced production of brain BDNF.

Example 14

Method of manufacturing IgG fusion proteins. The transfection of aeukaryotic cell line with immunoglobulin G (IgG) genes generallyinvolves the co-transfection of the cell line with separate plasmidsencoding the heavy chain (HC) and the light chain (LC) comprising theIgG. In the case of a IgG fusion protein, the gene encoding therecombinant therapeutic protein may be fused to either the HC or LCgene. However, this co-transfection approach makes it difficult toselect a cell line that has equally high integration of both the HC andLC-fusion genes, or the HC-fusion and LC genes. The preferred approachto manufacturing the fusion protein is the production of a cell linethat is transfected with a single plasmid DNA that contains all therequired genes on a single strand of DNA, including the HC-fusionprotein gene, the LC gene, the selection gene, e.g. neo, and theamplification gene, e.g. the dihydrofolate reductase gene. As shown inthe diagram of the fusion protein tandem vector in FIG. 12 , theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

Example 15

Construction and Use of GDNF-immunoglobulin fusion proteins.Glial-derived neurotrophic factor (GDNF) is a potent neuroprotectiveneurotrophin (Lin et al, Science 260:1130-1132 (1993)). GDNF could bedeveloped as a neuroprotective drug for acute conditions, such as acuteischemic stroke, or chronic conditions, such as Parkinson's disease, ormotor neuron disease, or post-stroke neural repair (Lapchak et al,Neuroscience 78:61-72 (1997); Kitagawa et al, Stroke 29:1417-1422(1998); Bohn, Exp. Neurol. 190:263-275 (2004); Kobayashi, et al. Stroke37:2361-2367 (2006)). However, GDNF, like other large molecule drugs,does not cross the blood-brain barrier (BBB) (Kastin et al, NeurosciLett. 340:239-241(2003)). Owing to the BBB problem, prior human clinicaltrials with recombinant GDNF delivered the protein to brain via invasivetrans-cranial routes, such as intra-cerebroventricular injection (Langet al, Ann Neurol. 59:459-466 (2006)) or convention enhanced diffusion(Patel et al, Ann. Neurol. 57:298-302 (2005)). However, both approacheswere limited by poor GDNF penetration into the brain, as well as adverseevents related to the invasive drug delivery system.

Large molecule drugs, such as GDNF, could be delivered to brainnon-invasively, via the trans-vascular route across the BBB with the useof molecular Trojan horse technology (Pardridge, Pharm. Res.27:1733-1744 (2007)). A BBB molecular Trojan horse is an endogenouspeptide, or receptor-specific peptidomimetic monoclonal antibody (MAb)that undergoes receptor-mediated transport across the BBB via endogenouspeptide receptors, such as the transferrin receptor or the insulinreceptor. Prior work has developed genetically engineered chimeric orhumanized MAb's to the human insulin receptor (HIR), as molecular Trojanhorses for the human brain (Boado et al, Biotechnol. Bioeng. 96:381-391(2007)). The non-transportable protein therapeutic is fused to the heavychain (HC) of the HIRMAb. It is essential that the geneticallyengineered fusion protein retain bi-functionality, and both bind withhigh affinity to the HIR, to cause BBB transport, and bind to thecognate receptor on brain cells, to induce the desired pharmacologiceffect, once the fusion protein penetrates the brain.

The present studies described the genetic engineering of a fusionprotein of the chimeric HIRMAb and human GDNF. The amino terminus ofmature human GDNF is fused to the carboxyl terminus of the HC of theHIRMAb (FIG. 28 ). Following expression in COS cells, thebi-functionality of the HIRMAb-GDNF fusion protein is evaluated withreceptor binding assays for both the HIR and the human GDNF receptor(GFR)-α1. The GDNF biologic activity of the fusion protein is alsoevaluated with bio-assays using 2 human neural cell lines, the SH-SY5Yline, and the SK-N-MC line. Finally, the in vivo neuroprotective effectsof the HIRMAb-GDNF fusion protein is demonstrated in rat brain using themiddle cerebral artery occlusion model (MCAO) of acute ischemic stroke.

Materials and Methods

Cloning of GDNF cDNA

The human prepro GDNF cDNA (GenBank P39905) corresponding to amino acidsMet¬¬1-Ile211 was cloned by the polymerase chain reaction (PCR) usingthe oligodexoynucleotides (ODNs) described in Table 6 and cDNA derivedfrom reverse transcription of polyA+RNA isolated from human U87 glialcells. DNA sequence analysis was performed and the nucleic acid andamino acid sequences of the cloned human prepro GDNF are given in SEQ IDNOs:43 and 44, respectively.

TABLE 6Oligodeoxynucleotide primers used in the RT-PCR cloning of human GDNF and inthe engineering of the HIRMAb-GDNF expression vectorHuman prepro GDNF FWD: phosphate-ATGAAGTTATGGGATGTCGTGGCTG (SEQ ID NO: 40)Human prepro GDNF REV: phosphate-TCAGATACATCCACACCTTTTAGCG (SEQ ID NO: 41)Mature human GDNF FWD: phosphate-CATCACCAGATAAACAAATGGCAGTG (SEQ ID NO: 42)

The GDNF cDNA was cloned by PCR using 25 ng polyA+RNA-derived cDNA, 0.2μM forward and reverse ODN primers (Table 6), 0.2 mMdeoxynucleosidetriphosphates, and 2.5 U PfuUltra DNA polymerase(Stratagene, San Diego, Calif.) in a 50 μl Pfu buffer (Stratagene). Theamplification was performed in a Mastercycler temperature cycler(Eppendorf, Hamburg, Germany) with an initial denaturing step of 95° C.for 2 min followed by 30 cycles of denaturing at 95° C. for 30 sec,annealing at 55° C. for 30 sec and amplification at 72° C. for 1 min;followed by a final incubation at 72° C. for 10 min. PCR products wereresolved in 0.8% agarose gel electrophoresis, and the expected majorsingle band of ˜0.6 kb corresponding to the human GDNF cDNA was produced(FIG. 29A). The human prepro GDNF cDNA was subcloned into the pcDNA3.1eukaryotic expression plasmid, and was designated pCD-GDNF. Theengineering of this plasmid was validated by DNA sequencing, and byexpression of immunoreactive GDNF in COS cells transfected withpCD-GDNF.

Engineering of HIRMAb-GDNF Expression Vector

For the engineering of the pHIRMAb-GDNF heavy chain (HC) expressionplasmid, the mature human GDNF cDNA (SEQ ID NO:51) corresponding toamino acids Ser¬78-Ile211 (SEQ ID NO:52) of full length human GDNF wascloned by PCR using the pCD-GDNF as template.

(SEQ ID NO: 51) TCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTG TGGATGTATCTGA (SEQ ID NO: 52) SPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI 

The PCR cloning reaction was performed as described above. The ODNs usedfor PCR are 5′-phosphorylated for direct insertion into the Hpal site ofthe pHIRMAb-HC expression plasmid (FIG. 29B). The pHIRMAb-HC plasmidencodes the HC of the chimeric HIRMAb, and dual transfection of COScells with this plasmid and a light chain (LC) expression plasmid,pHIRMAb-LC, allows for transient expression of either the chimericHIRMAb, or a fusion protein, in COS cells. The mature human GDNF forward(FWD) PCR primer (Table 6) introduces “CA” nucleotides to maintain theopen reading frame and to introduce a Ser-Ser linker between thecarboxyl terminus of the CH3 region of the HIRMAb HC and the aminoterminus of the GDNF. The fusion of the GDNF monomer to the carboxylterminus of each HC is depicted in FIG. 28 . This design stericallyrestricts the GDNF to a dimeric configuration, which replicates thenative dimeric conformation of GDNF that binds to the GFRα1 (Xu et al,1998; Eketjall et al, 1999). The GDNF reverse (REV) PCR primerintroduces a stop codon, “TGA,” immediately after the terminalisoleucine of the mature GDNF protein, and it is identical to the humanprepro GDNF REV ODN primer (Table 6) used in the cloning of the humanprepro GDNF cDNA. The engineered pHIRMAb-GDNF expression vector wasvalidated by DNA sequencing. The nucleic acid and amino acid sequencesfor fusion heavy chain (SEQ ID NOs 45 and 46) are as follow:

(SEQ ID NO: 45)ATGGACTGGACCTGGAGGGTGTTCTGCCTGCTTGCAGTGGCCCCCGGAGCCCACAGCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAACTACGATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACTTCTGAGAAATCTGCAGTCTATTTCTGTGCAAGAGAGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAGTTCATCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGA(SEQ ID NO: 46)MDWTWRVFCLLAVAPGAHSQVQLQQSGPELVKPGALVKISCKASGYTFTNYDIHWVKQRPGQGLEWIGWIYPGDGSTKYNEKFKGKATLTADKSSSTAYMHLSSLTSEKSAVYFCAREWAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSSSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCIAlso shown in FIG. 36.

The HIRMAb HC and LC cDNA expression cassettes are driven by thecytomegalovirus (CMV) promoter and contain the bovine growth hormone(BGH) polyadenylation (pA) sequence (FIG. 29B). The engineering of theuniversal pHIRMAb-HC vector was performed by insertion of a single Hpalsite at the end of the HIRMAb HC CH3 open reading frame (orf) by sitedirected mutagenesis (SDM).

Transient Expression of HIRMAb-GDNF Fusion Protein in COS Cells

COS cells were dual transfected with pHIRMAb-LC and pHIRMAb-HC-GDNFusing Lipofectamine 2000, with a ratio of 1:2.5, ug DNA:uLLipofectamine. Following transfection, the cells were cultured inserum-free VP-SFM (Invitrogen, Carlsbad, Calif.). The conditioned serumfree medium was collected at 3 and 7 days. The fusion protein waspurified by protein A affinity chromatography.

Human IgG and GDNF ELISAs

Human IgG ELISA was performed in Immulon 2 high binding plates (DynexTech., Chantilly, Va.) with COS cell conditioned medium. A goatanti-human IgG primary antibody (Zymed-Invitrogen, Carlsbad, Calif.) wasplated in 0.1 M NaHCO3 (100 μl, 2 μg/ml) and incubated for overnight at4 C. Plates were washed 0.01 M Na₂HPO4/0.15 M NaCl/pH=7.4/0.05% Tween-20(PBST), and blocked with 1% gelatin in PBST for 30 min at 22° C. Plateswere incubated with 100 μL/well of either human IgG1 standard or thefusion protein for 60 minutes at room temperature (RT). After washingwith PBST, a goat anti-human kappa LC antibody conjugated to alkalinephosphatase (Sigma Chemical Co., St. Louis, Mo.) was plated for 60 minat 37° C. Color development was performed with p-nitrophenyl phosphate(Sigma) at pH=10.4 in the dark. The reaction was stopped with NaOH, andabsorbance at 405 nm was measured in a BioRad ELISA plate reader. HumanGDNF immunoreactivity in COS cell conditioned medium was measured withthe double antibody sandwich GDNF E max Immunoassay System kit fromPromega (Madison, Wis.).

SDS-PAGE and Western Blotting

The homogeneity of protein A purified fusion protein produced by COScells was evaluated with a reducing 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomasie Bluestaining For Western blotting, immunoreactivity was tested with aprimary rabbit antibody to human GDNF (Santa Cruz Biotechnology, SantaCruz, Calif.) or a primary goat antiserum against human IgG heavy andlight chains (Vector Labs, Burlingame, Calif.).

HIR Receptor Assay

The affinity of the fusion protein for the HIR extracellular domain(ECD) was determined with an ELISA using the lectin affinity purifiedHIR ECD. CHO cells transfected with the HIR ECD were grown in serum freemedia (SFM), and the HIR ECD was purified with a wheat germ agglutininaffinity column. The HIR ECD (0.2 μg/well) was plated on Immulon 2 highbinding 96-well plates, and the binding of the chimeric HIRMAb, theHIRMAb-GDNF fusion protein, or human IgG1 to the HIR ECD was detectedwith a biotinylated goat anti-human IgG (H+L) antibody (0.3 μg/well),and the ABC Elite detection system (Vector Labs). The concentration thatcaused 50% binding to the HIR ECD, the ED50, was determined bynon-linear regression analysis using the WinNonlin software.

GFRα1 Receptor Assay

Binding of recombinant GDNF (Peproatech, Rocky Hill, N.J.), the chimericHIRMAb, or the HIRMAb-GDNF fusion protein to the ECD of recombinanthuman GFRα1 was measured with an ELISA. A mouse anti-human (MAH) IgG(Zymed-Invitrogen) was plated in 96-well plates overnight at 2 μg/well.Following aspiration, washing, and blocking with 1% bovine serum albumin(BSA), the Fc fusion of the human GFRα1 ECD (R&D Systems, Minneapolis,Minn.) was plated at 0.4 μg/well. Following an incubation at roomtemperature (RT) for 60 min, the wells were washed with PBST, and theGDNF, antibody, or fusion protein was plated for 2 hours at RT.Following washing in PBST, a goat anti-GDNF antibody (R&D Systems) wasplated at 0.4 ug/well for 30 min at RT. Following washing in PBST, aconjugate of alkaline phosphatase (AP) and a rabbit anti-goat (RAG)IgG(H+L) (Vector Labs) was plated and detection at 405 nm was performedwith an ELISA plate reader after color development withpara-nitrophenylphosphate (Sigma Chemical Co.).

SK-N-MC Bio-Assay

Human neural SK-N-MC cells were dual transfected with the c-ret kinaseand a luciferase reporter plasmid under the influence of the 5′-flankingsequence (FS) of the rat tyrosine hydroxylase (TH) gene. The cells weregrown in collagen coated 24-well dishes to 70% confluency in Dulbecco'smodified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 400μg/mL G418. To begin the assay, the medium was aspirated, and 400μL/well of fresh DMEM/10% FBS was added along with either recombinantGDNF or fusion protein. After a 24 hour incubation at 37° C., the wellswere aspirated, and the cells extracted with 200 μL/well of LuciferaseReporter Lysis buffer (Promega, Madison, Wis.). Followingcentrifugation, luciferase enzyme activity was measured in the lysatewith a luminometer, and luciferase enzyme activity, reported as picogram(pg) of luciferase, was normalized per mg sample protein using thebicinchoninic acid (BCA) protein assay (Pierce Chemical Co., Rockford,Ill.).

SH-SY5Y Bio-Assay

Human neural SH-SY5Y cells were plated in 96 well plates in DMEM/Ham F12(1:1) in 10% FBS and grown until the cells reached 70% confluency. Themedium was supplemented with 10 μM retinoic acid to inducedifferentiation of the cells over a 10 day period. After celldifferentiation, the medium was changed, and supplemented with eitherGDNF or HIRMAb-GDNF fusion protein at zero time, and cell proliferationwas measured over the 5 days with the CellTiter 96® AQueousNon-Radioactive Cell Proliferation Assay (Promega), which uses thetetrazolium compound,(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS), and the electron coupling reagent, phenazinemethosulfate (PMS). Absorbance at 490 nm is a measure of formazanproduction, which is proportional to the number of viable cells.

Middle Cerebral Artery Occlusion Model

The permanent middle cerebral artery occlusion (MCAO) model wasperformed as described previously in adult Sprague-Dawley rats (Zhangand Pardridge, Brain Res. 889:49-56 (2001)). The right middle cerebralartery of the anesthetized rat was occluded by insertion of anintra-luminal 4-0 nylon suture; the suture was coated in 0.1%poly-L-lysine and dried at 60° C. for 1 hour. Within 15 minutes of theocclusion, the rat was treated with an intra-cerebral injection ofeither HIRMAb-GDNF fusion protein (130 μg in 10 μL) or an equal volumeof saline. Since the fusion protein is 17% GDNF, the dose of fusionprotein is equivalent to a dose of GDNF of 22 μg, which is a dose thatcauses pharmacological effects in the brain (Sullivan et al, 1998). Thedrug was injected into the brain under stereotaxic guidance with thefollowing coordinates: 0.2 mm posterior to bregma, 4 0 mm lateral to themidline, and 5.0 mm deep from the dural surface. The arterial nylonsuture was left in place for permanent occlusion of the middle cerebralartery. A total of 20 rats were used for the study; 9 rats (5 treatedwith saline and 4 treated with HIRMAb-GDNF) expired prematurely and wereexcluded from the study. The rats were allowed to recover, and wereeuthanized 24 hours later, and coronal sections of brain were preparedwith a rat brain matrix (ASI Instruments, Warren, Mich.), followed bystaining with 2% 2,3,5-triphenyltetrazolium chloride (TTC), as describedpreviously (Zhang and Pardridge, Brain Res. 889:49-56 (2001)). TTCstains healthy brain red, and infarcted brain is colorless. The stainedbrain sections were scanned on a UMAX PowerLook II flatbed scanner, andthe area of the hemispheric infarct volumes was determined with the NIHImage software. The volume of the infarct was computed from the area ofthe infarcted zone and length (2 mm) of the coronal slices of brainhemisphere (Zhang and Pardridge, Brain Res. 889:49-56 (2001)). Thescanned color image was converted to an inverted grayscale image inPhotoshop, so that infarcted brain appears black and healthy brainappears white.

The neurologic deficit was determined 24 hours after the occlusion, asdescribed previously (Zhang and Pardridge, Stroke 32:1378-1384 (2001)),and scored as follows: 0, no deficit; 1, failure to extend contralateralforepaw fully; 2, decreased grip of contralateral forelimb while tail ispulled; 3, spontaneous circling to left; 4, walks only when stimulatedwith decreased level of consciousness.

Results

DNA sequencing of the expression cassette of the pCD-GDNF encompassed1,752 nucleotides (nt), including a 715 nt CMV promoter, a 636 nt preproGDNF open reading frame, and a 401 nt BGH sequence, which predicted a211 amino acid human preproGDNF protein, including a 19 amino acidsignal peptide with 100% identity with the known sequence for human GDNF(P39905). Transfection of COS cells with pCD-GDNF resulted in highlevels of immunoreactive GDNF the medium at 3 and 7 days followingtransfection.

The cDNA corresponding to the 134 amino acid mature GDNF was amplifiedby PCR using the ODNs in Table 6 and the pCD-GDNF as template, and thiscDNA was subcloned into the Hpal site of the pHIRMAb-HC plasmid, asoutlined in FIG. 29B. DNA sequencing of the expression cassette of thepHIRMAb-GDNF plasmid encompassed 2,890 nt, including a 714 nt CMVpromoter, a 9 nt full Kozak site (GCCGCCACC), a 1,797 nt HIRMAb HC-GDNFfusion protein open reading frame, and a 370 nt BGH sequence. Theplasmid encoded for a 598 amino acid protein, comprised of a 19 aminoacid IgG signal peptide, the 443 amino acid HIRMAb HC, a 2 amino acidlinker (Ser-Ser), and the 134 amino acid human GDNF minus the signalpeptide or propeptide. The predicted molecular weight of the heavy chainfusion protein, minus glycosylation, is 63,860 Da, with a predictedisoelectric point (pI) of 9.03.

Dual transfection of COS cells with the pHIRMAb-GDNF and the pHIRMAb-LCresulted in medium human IgG levels of 2.0 μg/mL, as determined with ahuman Fc specific ELISA. The level of immunoreactive GDNF in the mediumwas measured with a GDNF-specific ELISA. The data from both the humanIgG and human GDNF ELISAs are given in Table 7, and the medium levelsare expressed as nM concentrations to allow for comparison of the 2assays.

TABLE 7 HIRMAb-GDNF fusion protein concentrations in the medium oftransfected COS cells following transfection with pHIRMAb-LC andpHIRMAb-GDNF 3 day 7 day Target of primary concentration concentrationantibody (nM) (nM) Anti-human IgG Fc 3.1 ± 0.2 11.3 ± 1.4 Anti-humanGDNF 2.0 ± 0.5 12.6 ± 3.1 Mean ± SE (n = 3 dishes per point). The mediumimmunoreactive human IgG or GDNF was <0.1 nM in dishes treated withLipofectamine 2000 alone.

As shown in Table 7, the concentration of the HIRMAb-GDNF fusion proteinthe medium was the same irrespective of whether the human IgG or GDNFELISA is used.

The HIRMAb-GDNF fusion protein was purified by protein A affinitychromatography. Following SDS-PAGE and Coomasie blue staining, the sizeof the light chain (LC) was the same for both the HIRMAb and theHIRMAb-GDNF fusion protein (FIG. 30 ). The size of the heavy chain (HC)of the fusion protein was about 15 kDa larger than the HC of the HIRMAb(FIG. 30 ). On Western blotting, the LC of either the HIRMAb or theHIRMAb-GDNF fusion protein reacted equally on the Western with a primaryantibody directed against the human IgG (H+L), as shown in FIG. 31 . Thesize of the HC of the fusion protein was about 15 kDa larger than thesize of the HC of the HIRMAb on both Western blots using either theanti-human IgG primary antibody (FIG. 31 ) or the anti-human GDNFprimary antibody (FIG. 31 ). The anti-GDNF primary antibody reacts withthe HC of the fusion protein, and with recombinant GDNF, but does notreact with the HIRMAb (FIG. 31 ).

The affinity of the fusion protein for the HIR extracellular domain(ECD) was determined with a ligand binding assay using lectin affinitypurified HIR ECD (Methods). There is comparable binding of either thechimeric HIRMAb or the HIRMAb-GDNF fusion protein for the HIR ECD withED50 of 0.50±0.11 nM and 0.87±0.14 nM, respectively (FIG. 32 ).

The affinity of either GDNF or the HIRMAb-GDNF fusion protein for theECD of the human GFRα1 was measured with an ELISA, which is outlined inFIG. 33A Human GDNF bound to the GFRal with an ED50 of 1.03±0.18 nM(FIG. 33B, top panel). The affinity of the HIRMAb-GDNF fusion proteinfor the GFRα1 was also high, with an ED50 of 1.68±0.17 nM (FIG. 33B,bottom panel). The biologic activity of GDNF or the HIRMAb-GDNF fusionprotein was also evaluated with bio-assays in human neural cell lines.The human neural SK-N-MC cell line was dual transfected with the c-retkinase, and with a luciferase expression plasmid under the influence ofthe 5′-flanking sequence (FS) of the rat TH promoter. Addition of GDNFto the medium activates the TH promoter via activation of theGFRα1/c-ret kinase system, and this leads to increased luciferaseexpression (FIG. 34A). GDNF increased luciferase expression with an ED50of 1.03±0.31 nM, and the HIRMAb-GDNF fusion protein also increasedluciferase expression with an ED50 of 1.68±0.45 nM (FIG. 34B). Both GDNFand the HIRMAb-GDNF fusion protein activated cell division of humanneural SH-SY5Y cells following retinoic acid differentiation, and bothproteins increased cell division about 50% over a 5 day incubationincubation period (Table 8).

TABLE 8 Enhanced proliferation of retinoic acid differentiated humanneural SH-SY5Y cells bv human GDNF or HIRMAb-GDNF Treatment A490 GDNF (3nM) 0.43 ± 0.02 ^(a) HIRMAb-GNDF (5 nM) 0.42 ± 0.03 ^(a) control 0.29 ±0.01 Mean ± SE (n = 6); ^(a) p <0.005. Cell proliferation measured withthe MTS assay as described herein.

The intra-cerebral injection of the HIRMAb-GDNF fusion protein caused a77% reduction in hemispheric stroke volume, from 331±33 mm3 to 77±22 mm3(mean±SE, n=5-6 rats per group), which was significant at the p<0.001level (Student's t-test), as shown in FIG. 35A. Similarly, the corticalstroke volume was reduced 86% from 241±26 mm3 to 34±9 mm3 (p<0.001), andthe sub-cortical stroke volume was reduced 52% from 90±10 mm3 to 43±16mm3 (p<0.025), as shown in FIG. 35A. A representative TTC stain of thebrain 24 hours after permanent MCAO and intra-cerebral injection ofeither saline or the HIRMAb-GDNF fusion protein is shown in FIGS. 35Band 35C, respectively. The neurologic deficit score was 3.0±0.7 and1.3±0.6 (mean±SE, n=5-6 rats per group) in the saline treated andHIRMAb-GDNF treated rats, respectively, and this improvement inneurologic deficit was significant at the p<0.05 level (Student'st-test).

Discussion

The results of this study were consistent with the followingconclusions. First, a bi-functional IgG-GDNF fusion protein wasgenetically engineered, wherein mature human GDNF was fused to thecarboxyl terminus of the heavy chain (HC) of a chimeric HIRMAb (FIG. 28), and expressed and secreted in COS cells (Table 8, FIGS. 30 and 31 ).Second, the HIRMAb-GDNF fusion protein was bi-functional and bound theHIR and human GFRα1 with high affinity (FIGS. 32 and 33 ). Third, theHIRMAb-GDNF fusion protein had activity in bio-assays of human neuralcells comparable to recombinant human GDNF (FIG. 34 , Table 8). Fourth,the HIRMAb-GDNF was neuroprotective in rat brain in vivo in thepermanent MCAO stroke model, and caused a marked reduction in strokevolume (FIG. 35 ).

GDNF is a member of the transforming growth factor (TGF)-α gene family,along with other neurotrophins, such as neuturin, persephin, or artemin.GDNF reacts with its cognate receptor, GFRα1, as a homo-dimer (Eketjallet al, 1999). Therefore, the GDNF fusion construct described in thiswork (FIG. 28 ) places GDNF in a dimeric configuration that mimicked theconformation of the neurotrophin at the receptor. The prepro GDNF couldbe fused to the amino terminus of the HIRMAb HC. However, this wouldinterfere with HIRMAb binding to the HIR, which would impair transportof the fusion protein across the BBB via the insulin receptor. Moreover,the mature GDNF protein folded into a biologically active conformation,despite the absence of the prepro GDNF peptide in the fusion proteindescribed in this work. The goal of fusion protein engineering was toretain the bi-functional characteristics of the fusion construct, andthis was accomplished in the case of the HIRMAb-GDNF fusion protein.

The fusion protein was secreted in high amounts to the medium by COScells co-transfected with the HC expression plasmid, pHIRMAb-GDNF (FIG.29B), and the LC expression plasmid, pHIRMAb-LC (Table 7). The HC of thefusion protein is about 15 kDa larger than the HC of the chimericHIRMAb, based on either SDS-PAGE gels with Coomassie blue staining (FIG.30 ), or on Western blot analysis using primary antibodies to eitherhuman IgG or human GDNF (FIG. 31 ). The anti-human IgG antibody reactsequally with the LC of the fusion protein or the chimeric HIRMAb, sinceboth proteins use the same LC. Both the HIRMAb-GDNF fusion protein andthe HIRMAb bind with high affinity to the HIR, and with comparableaffinity (FIG. 32 ).

The HIRMAb-GDNF fusion protein retains high affinity binding to thehuman GFRα1 receptor, and the affinity constant of binding is comparableto that of recombinant GDNF (FIG. 33B). The high affinity binding of theHIRMAb-GDNF fusion protein to the GFRα1 was translated into biologicalactivity in two different human neural cell lines. The SH-SH5Y cell lineexpresses the GFRα11, but does not express the c-ret kinase in theundifferentiated state (Xiao et al, J. Neurochem. 82:701-808 (2002)).Both the GFRα1 and the c-ret kinase must be expressed to enable GDNFactivation of a neural cell (Cik et al, 2000). However, differentiationof SH-SH5Y cells by retinoic acid causes an induction of the expressionof the c-ret kinase (Xiao et al, J. Neurochem. 82:701-808 (2002)).Following differentiation by retinoic acid, the SH-SY5Y cellsdemonstrate enhanced proliferation in response to either recombinanthuman GDNF or the HIRMAb-GDNF fusion protein (Table 8). The SK-N-MChuman neural cell line also expresses the GFRα1 receptor, but not thec-ret kinase (Hirata and Kiuchi, Brain Res. 983:1-12 (2003)). However,following co-transfection of these neural cells with a c-ret kinase cDNAand a plasmid encoding luciferase under the influence of 2 kb of the5′-FS of the rat TH promoter, these cells respond to GDNF (Tanaka et al,Brain res. Brain Res. Protoc. 11:119-122 (2003)). As outlined in FIG.34A, the extracellular GDNF binds the GFRα1, which activates the c-retkinase, which induces a signal transduction cascade leading toactivation of the TH promoter. Accordingly, both recombinant GDNF andthe HIRMAb-GDNF fusion protein caused an approximate 500% increase incellular luciferase enzyme activity. The GDNF ED50 in the luciferaseassay is 1.0±0.1 nM, and the HIRMAb-GDNF ED50 in this assay is 1.7±0.5nM (FIG. 34B). These ED50 values are identical to the ED50 of saturablebinding of either GDNF or the HIRMAb-GDNF fusion protein in the GFRα1binding assay (FIG. 33B). This correlation indicates that therate-limiting step in activation of neural pathways by GDNF is bindingto the cell membrane GFRα1 receptor.

The intra-cerebral injection of the HIRMAb-GDNF fusion protein in ratbrain following permanent middle cerebral artery occlusion (MCAO)resulted in a 77% reduction in hemispheric stroke volume from 331±33 mm³to 77±22 mm3 (p<0.001), as shown in FIG. 35A. The neuroprotection wasboth cortical and sub-cortical, as the cortical stroke volume wasreduced 86% from 241±26 mm³ to 34±9 mm3 (p<0.001), and the sub-corticalstroke volume is reduced 52% from 90±10 mm3 to 43±16 mm3 (p<0.025) (FIG.35A). A representative TTC stain of the rat brain at 24 hours after thepermanent MCAO is shown in FIG. 35B, for the saline treated rat, and inFIG. 35C for the HIRMAb-GDNF fusion protein treated rat. The reductionin stroke volume was correlated with a functional improvement as theneurologic deficit was reduced from 3.0±0.3 to 1.3±0.6 (p<0.05) in therats treated with the HIRMAb-GDNF fusion protein as compared to thesaline treated rats. These findings of in vivo neuroprotection of theHIRMAb-GDNF fusion protein correlated with the tissue culture bio-assays(Table 8, FIG. 34B), and confirmed the neuroprotective effects of theHIRMAb-GDNF fusion protein on neural cells in brain. The MCAO findingsconfirmed prior work showing the beneficial effects of intra-cerebralGDNF in acute stroke (Kitagawa et al, 1998). Recent work showed thatchronic treatment of the brain with intra-cerebral GDNF may promotestriatal neurogenesis following stroke (Kobayashi et al, Stroke37:2361-2367 (2007)). Irrespective of whether the brain is treatedacutely or chronically with GDNF, the neurotrophin had to bere-engineered to cross the BBB before clinical trials in human strokecan be initiated.

The HIRMAb-GDNF fusion protein could not be delivered to rat brain inthe MCAO model following intravenous administration, because the HIRMAbpart of the fusion protein is not reactive with the rodent insulinreceptor. However, the HIRMAb is active in Old World primates, such asthe Rhesus monkey (Pardridge et al, Pharm. Res. 12:807-816 (1995)).Recent work has shown that fusion proteins of the HIRMAb andbrain-derived neurotrophic factor (Boado et al, Biotechnol. Bioeng.97:1376-1386 (2007)), a single chain Fv antibody (Boado et al, BioconjugChem. 18:447-455 (2007)), or iduronidase, a lysosomal enzyme (Boado etal, Biotechnol Bioeng. 99: 475-484 (2008)), all are rapidly transportedacross the Rhesus monkey BBB in vivo. In the primate brain, the uptakeof the fusion protein is approximately 1% of the injected dose (ID)(Boado et al, Biotechnol Bioeng. 99: 474-484 (2008)). Since the weightof the human brain is about 10-fold greater than the weight of theRhesus monkey brain, it is expected that the brain uptake of theHIRMAb-GDNF fusion protein by the human brain will be about 0.1% of theID. Given an injection dose of 25 mg, the expected brain concentrationof the fusion protein is about 25 ng/gram human brain, which isequivalent to 5 ng/gram of GDNF, since the fusion protein is about 20%GDNF and 80% HIRMAb. The concentration of GDNF in the human brain is0.2-1 ng/gram (Wiesenhofer et al, Acta Neuropathol. (Berl) 99:131-137(2000)). Therefore, the dosing of 25 mg of the HIRMAb-GDNF fusionprotein to a 60 kg human could result in pharmacologically significantincrease in GDNF in the brain. In experimental Parkinson's disease,pharmacological effects are achieved with just a 3-fold increase inbrain GDNF concentration (Eslamboli et al, J. Neurosci. 25:769-777(2005)). Aberrant neuronal sprouting is induced when the brain GDNFconcentration is increased >100-fold (Eslamboli et al, J. Neurosci.25:769-777 (2005)).). However, such large increases in brain GDNF willnot be produced by therapeutic dosing of the HIRMAb-GDNF fusion proteinin humans.

In conclusion, these studies described the genetic engineering,transient expression in COS cells, and validation of a HIRMAb-GDNFfusion protein, which represents a re-engineering of this neurotrophinto enable transport across the human BBB in vivo. The fusion protein canbe administered by systemic injection to humans for treatment ofmultiple neurologic disorders, including stroke, neural repair,Parkinson's disease, or motor neuron disease.

Example 16

Expression of HIRMAb-GDNF Fusion Protein Following Stable Transfectionof CHO Cells

A tandem vector (TV) encoding the HIRMAb-GDNF fusion protein wasengineered as shown in FIG. 37 , and is designated HIRMAb-GDNF TV. TheTV contains on a single piece of DNA the fusion heavy chain (HC), thelight chain (LC), and the gene for DHFR. DNA sequencing of the entire6,300+ nucleotides (nt) of the HIRMAb-GDNF TV using customoligodeoxynucleotides (ODNs) showed the sequence was comprised of 6,342nt (SEQ ID NO. 47), which included the following domains.

-   -   731 nt cytomegalovirus (CMV) promoter    -   9 nt Kozak sequence (GCCGCCACC)    -   705 nt open reading frame (orf) encoding the HIRMAb LC    -   291 nt bovine growth hormone (BGH) polyA (pA) sequence    -   23 nt linker    -   714 nt CMV promoter    -   9 nt Kozak sequence    -   1,797 ORF encoding the fusion gene of the HIRMAb HC and GDNF    -   296 nt BGH pA    -   254 SV40 promoter    -   9 nt Kozak sequence    -   564 murine DHFR orf    -   940 hepatitis B virus (HBV) pA

The HIRMAb-GDNF TV also included the expression cassette encoding neo,the neomycin resistance gene, to enable selection with G418 (FIG. 37 ).It was necessary to include the HC fusion gene, the LC gene, and theDHFR gene on a single piece of DNA, or tandem vector (FIG. 37 ) to allowfor equally high expression of all 3 genes in the transfected CHO cell.

The HIRMAb-GDNF TV sequence encoded for a 579 amino acid (AA) HC fusionprotein (SEQ ID NO 48), which was comprised of a 19 AA IgG signalpeptide, the 443 AA HIRMAb HC, a 2 AA linker, and the 134 AA human GDNF.The predicted molecular weight (MW) of the non-glycosylated HC was63,860 Daltons (Da) and the predicted isolectric point (pI) of thefusion HC protein was 9.03. The HIRMAb-GDNF TV sequence encoded for a234 AA LC protein (SEQ ID NO 49), which was comprised of a 20 AA IgGsignal peptide, and the 214 AA HIRMAb LC. The predicted MW of the LC was23,398 Da and the predicted pI of the LC protein was 5.45. TheHIRMAb-GDNF TV sequence encoded for a DHFR protein (SEQ ID NO 50), thathad an AA sequence 100% identical with the known AA sequence of murineDHFR. The domain structure of the HC is shown in FIG. 36 and the domainstructure of the LC is shown in FIG. 38 .

Electroporation of CHO Cells.

DG44 CHO cells were grown in serum-free medium (SFM), containing 1× HTsupplement (hypoxanthine and thymidine). DG44 CHO cells (5×10⁶ viablecells) were electroporated with 5 μg PvuI-linearized TV plasmid DNA. Thecell-DNA suspension is then incubated for 10 min on ice. Cells areelectroporated with pre-set protocol for CHO cells, i.e., square wavewith pulse of 15 msec and 160 volts. After electroporation, cells areincubated for 10 min on ice. The cell suspension is transferred to 50 mlculture medium and plated at 125 μl per well in 40×96-well plates(10,000 cells per well), and 4,000 wells per study.

Selection and Amplification with Methotrexate and Screening IgG ELISA.

Following electroporation (EP), the CHO cells were placed in theincubator at 37° C. and 8% CO₂. Owing to the presence of the neo gen inthe TV, transfected cell lines are initially selected with G418. The TValso contains the gene for DHFR, so the transfected cells were alsoselected with 20 nM methotrexate (MTX) and HT-deficient medium. Oncevisible colonies are detected at about 21 days after EP, the conditionedmedium was sampled for human IgG by ELISA. Plates were removed from theincubator and transferred to the sterile hood where 100 μL samples weretaken from each well using a Precision Pipettor system and transferredinto a sterile 96-well tissue culture plate, which was then used for thehuman IgG ELISA. The media taken from the EP plates for the ELISA wasreplaced with 100 μL of SFM and cells were returned to the incubator at37° C. and 8% CO₂. Wells with high human IgG signals in the ELISA weretransferred from the 96-well plate to a 24-well plate with 1 mL of SFM.The 24-well plates were returned to the incubator at 37° C. and 8% CO₂.The following week IgG ELISA was performed on the clones in the 24-wellplates. This was repeated through the 6-well plates to T75 flasks andfinally to 60 mL and 125 mL square plastic bottles on an orbital shaker.After the cells adapted to the 60 mL bottle on the orbital shaker at 120RPM they were transferred into a 125 mL plastic square bottle with 12 mLof SFM. At this stage, the final MTX concentration was 80 nM, and themedium IgG concentration, which is a measure of HIRMAb-GDNF fusionprotein in the medium was >10 mg/L at a cell density of 10⁶/mL.

Dilutional Cloning of CHO Cells.

Clones selected for dilutional cloning (DC) were removed from theorbital shaker in the incubator and transferred to the sterile hood. Thecells were diluted to 500 mL in F-12K medium with 5% dialyzed fetalbovine serum (d-FBS) and penicillin/streptomycin, and the final dilutionwas 8 cells per mL, so that 4,000 wells in 40×96-well plates were platedat a cell density of 1 cell per well (CPW). After the cell suspensionwas prepared, within the sterile hood, a 125 μL aliquot was dispensedinto each well of a 96-well plate using an 8-channel pipettor or aPrecision Pipettor system. The plates were then returned to theincubator at 37° C. and 8% CO₂. The cells diluted to 1 cell/well cannotsurvive without serum. On day 6 or 7, DC plates were removed from theincubator and transferred to the sterile hood where 125 μL of F-12Kmedium with 5% dialyzed fetal bovine serum (d-FBS) was added to eachwell. After this step, the selection media contained 5% d-FBS, 30 nM MTXand 0.25 mg/mL Geneticin. On day 21 after the initial 1 CPW plating,aliquots from each of the 4,000 wells were removed for human IgG ELISA,using robotics equipment. DC plates were removed from the incubator andtransferred to the sterile hood, where 100 μL of media was removed perwell of the 96-well plate and transferred into a new, sterile sample96-well plate using an 8-channel pipettor or a Precision Pipettorsystem.

ELISA Screening of 4,000 Wells.

On day 20 after the initial 1 CPW plating, 44 96-well Immunoassay plateswere plated with 100 μL of 1 μg/mL solution of Primary antibody, a mouseanti-human IgG in 0.1M NaHCO3. Plates were incubated overnight in the 4°C. Revco refrigerator. The following day, the plates were washed with 1×TBST 5 times, and 100 μL of 1 μg/mL solution of secondary antibody andblocking buffer were added. Immulon plates were washed with 1× TBST 5times. 100 μL of 1 mg/mL of 4-nitrophenyl phosphatedi(2-amino-2-ethyl-1,3-propanediol) salt in 0.1M glycine buffer wereadded to the Immulon 96-well Immunoassay plates using the BioTek uFill.Plates were read on a microplate reader. The assay produced IgG outputdata for 4,000 wells/experiment. The highest producing 24-48 wells wereselected for further propagation.

Adaptation of Cloned Cells to Serum-Free Medium (SFM).

The highest producing 24-well plates from the 1 CPW DC transferred tothe sterile hood were gradually subcloned through 6-well dishes, T75flasks, and 125 mL square plastic bottles on an orbital shaker. Duringthis process the serum was reduced to zero, at the final stage ofcentrifugation of the cells and resuspension in SFM.

Second Round Dilutional Cloning.

The above procedures were repeated with a second round of dilutionalcloning, again at 0.5 cells/well (CPW). At this stage, approximately 40%of the wells showed any cell growth, and all wells showing growth alsosecreted human IgG. These results confirmed that on average only 1 cellis plated per well with these procedures, and that the CHO cell lineoriginated from a single cell.

Downstream Processing and 3-Column Purification of the HIRMAb-GDNFFusion Protein.

Following the second round of dilutional cloning, the highest producingcell line secreting the HIRMAb-GDNF fusion protein was propagated inserum-free medium to a total volume of 2,000 mL in several 1 L squareplastic bottles on an orbital shaker. The HIRMAb-GDNF fusion protein waspurified from the CHO cell conditioned medium using the followingdown-stream processing:

-   -   Depth filtration with a 0.2 m² 0.65 um GF filter in series with        an 0.05 m² 0.2 μm ultrafilter    -   Volume reduction to 400 mL using a Pellicon-2 tangential flow        filtration (TFF) system    -   Ultra-filtration with a 0.2 mm ultra-filter and application to a        7 mL column of protein A. Following application to the column,        the column was eluted with 1 M NaCl, which elutes DNA        non-specifically absorbed to the column, and the product is        eluted as a single peak with 0.1 M sodium acetate/pH=3.7. The        acid eluate was neutralized with 1 M Tris base and concentrated        to 5 mL.    -   Cation exchange (CATEX) chromatography in bind-elute mode was        performed with a 5 mL column of SP Sepharose FF equilibrated        with 0.02 M MES and 0.05 M NaCl. The conductivity of the sample        was reduced to <5 mS/cm prior to application to the column. The        column was successively eluted with step gradients of 0.02 M        MES/pH=5.5 containing 0 25 M NaCl, 0.35 M NaCl, 0.5 M NaCl, and        1M NaCl. The HIRMAb-GDNF fusion protein eluted in 0.5 M NaCl.    -   Anion exchange (ANEX) chromatography in flow-through mode was        performed with a 5 mL column of Q Sepharose FF equilibrated with        0.025 M MES/pH=5.5 and 0.05 M NaCl. The conductivity of the        sample was reduced to <7 mS/cm. The HIRMAb-GDNF fusion protein        eluted in the flow-through.

Biochemical Characterization of 3-Column Purified CHO Cell DerivedHIRMAb-GDNF Fusion Protein.

The purity and potency of the CHO derived HIRMAb-GDNF fusion protein wasassessed with the following procedures:

-   -   (a) SDS-PAGE. The HIRMAb-GDNF fusion protein was purified to        homogeneity based on reducing and non-reducing sodium dodecyl        sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A        characteristic “fingerprint” of the HIRMAb-GDNF fusion protein        is the detection of 3 heavy chain glycoforms, which arise from        differential glycosylation of the GDNF part of the fusion        protein. This pattern is reproducible from run to run.    -   (b) GDNF and human IgG Western blot. The CHO derived HIRMAb-GDNF        fusion protein was electrophoresed on a 12% SDS-PAGE gel and        blotted to nitrocellulose for Western blotting with primary        antibodies to either human IgG (left panel, FIG. 39 ), or to        human GDNF (right panel, FIG. 39 ). Both the anti-human IgG        antibody and the anti-human GDNF antibody reacted specifically        with the heavy chain of the HIRMAb-GDNF fusion protein.    -   (c) Human insulin receptor (HIR) binding of AGT-190. The        extracellular domain (ECD) of the HIR was purified by lectin        affinity chromatography from serum free medium conditioned by        CHO cells that were permanently transfected with the HIR ECD .        The HIR ECD was plated in ELISA wells to bind the chimeric        HIRMAb without GDNF fused and the HIRMAb-GDNF fusion protein. As        shown in FIG. 40 , the affinity of the CHO-derived HIRMAb-GDNF        fusion protein binding to the HIR is high, and not significantly        different from the binding of the chimeric HIRMAb. These data        indicate the affinity of the HIRMAb for the HIR is not affected        by the fusion of GDNF to the carboxyl terminus of the IgG. The        binding constant (ED50) shown in FIG. 40 was determined by        non-linear regression analysis of the binding isotherm.    -   (d) GFRa1 binding of the HIRMAb-GDNF fusion protein. The        affinity of either GDNF or the HIRMAb-GDNF fusion protein for        the GDNF receptor, GFRal, was determined with a specific ELISA        format, which is outlined in FIG. 33 . In this assay, GDNF binds        with high affinity for the GFRal extracellular domain (ECD), and        the affinity of the CHO-derived HIRMAb-GDNF fusion protein for        the GFRa1 (ED50=1.44±0.07 nM) was comparable to GDNF        (ED50=1.00±0.09 nM).    -   (e) GDNF bio-assay in rat brain in vivo. The intra-cerebral        injection of GDNF in experimental stroke results in        neuroprotection, as reflected in a reduced stroke volume.        Therefore, the permanent middle cerebral artery occlusion (MCAO)        model was used as a rapid in vivo bio-assay of CHO-derived        HIRMAb-GDNF fusion protein neuroprotective properties in vivo,        similar to that shown for the COS derived fusion protein (FIG.        35 ). The intra-cerebral injection of the HIRMAb-GDNF fusion        protein caused a 74% reduction in hemispheric stroke volume,        from 338±33 mm³ to 87±27 mm³ (mean±SE, n=5-6 rats per group),        which was significant at the p<0.001 level (Student's t-test).        Similarly, the cortical stroke volume was reduced 80% from        238±25 mm³ to 48±18 mm³ (p<0.001), and the sub-cortical stroke        volume was reduced 39% from 101±10 mm³ to 39±11 mm³ (p<0.025).        The neurologic deficit score was 3.6±0.2 and 1.4±0.7 (mean±SE,        n=5-6 rats per group) in the saline treated and CHO-derived        HIRMAb-GDNF fusion protein treated rats, respectively, and this        improvement in neurologic deficit was significant at the p<0.05        level.    -   (f) Size exclusion high performance liquid chromatography. The        absence of aggregates in the purified HIRMAb-GDNF fusion protein        was demonstrated with size exclusion chromatography (SEC) high        performance liquid chromatography (HPLC) using a G3000 SWXL        column, 0.78×30 cm and an HPLC pump at 0.5 mL/min with detection        at 280 nm. As shown in FIG. 41 , the CHO-derived HIRMAb-GDNF        fusion protein elutes as a single peak, removed from the void        volume, with no detectable aggregates. The absence of aggregates        is also demonstrated by the native polyacrylamide gel        electrophoresis (FIG. 42 ).    -   (g) Carbohydrate analysis. The CHO-derived HIRMAb-GDNF fusion        protein was analyzed for neutral monosaccharide content and for        the N-terminal oligopeptide fingerprint. The fusion protein is        glycosylated at 3 different N-linked asparagine sites, including        1 site within the CH2 region and 2 sites within the GDNF region        of the fusion protein heavy chain The neutral monosaccharide        content (moles monosaccharide/mole fusion protein) was 7.7 for        N-acetyl glucosamine, 5.3 for mannose, 2.3 for galactose, 1.6        for fucose, and 0.19 for N-acetyl galactosamine. These 4 sugars        are organized as 1 of 4 different terminal structures,        designated G0, G1(1,6), G1(1,3), or G2, depending on whether        there is 0, 1, or 2 terminal galactose moieties, respectively.        The profile for AGT-190 is G0>G1>G2, which is typical of        CHO-derived recombinant proteins, and indicative of relative        reduced terminal galactosylation. The glycosylation of        HIRMAb-GDNF fusion protein on at least 3 different asparagine        residues explains the heterogeneity of the heavy chain on        Western blotting (FIG. 39 ).    -   (h) Binding to human blood-brain barrier. Capillaries, which        form the BBB in vivo, were isolated from human autopsy brain in        a purified preparation as shown by light microscopy (FIG. 43 ).        The HIRMAb-GDNF fusion protein was radio-labeled by tritiation,        and a radio-receptor assay was performed to demonstrate binding        of the labeled HIRMAb-GDNF fusion protein to the HIR at the        human BBB, using the isolated human brain capillaries. There was        a high level of binding of the HIRMAb-GDNF fusion protein to the        human brain capillary and the binding was displaced by unlabeled        murine HIRMAb (FIG. 43B).

Example 17

Treatment of Parkinson's Disease with the HIRMAb-GDNF Fusion Protein

Parkinson's disease (PD) is a neurodegenerative condition that affectsthe dopaminergic neurons of the nigral-striatal tract. GDNF is a potenttrophic factor for these dopaminergic neurons. The intra-cerebralinjection of the GDNF protein into the brain of rats with experimentalPD can protect these neurons, and blocks further axotomy of the fibersprojecting from the substantia nigra to the striatum. Accordingly,recombinant human GDNF has been developed as a new therapeutic for PD.However, GDNF does not cross the BBB. Therefore, GDNF was administeredvia a trans-cranial route using intra-cerebroventricular (ICV)injection. However, GDNF delivered by ICV injection was found to notpenetrate into the brain parenchyma and was ineffective in patients withPD, and this delivery approach was abandoned. Subsequently, GDNF hasbeen administered by convection enhanced diffusion (CED). In thisapproach, a reservoir holding the GDNF is implanted in the abdomen, anda catheter is run through the skin from the reservoir to the brain via atrans-cranial passage. The reservoir has a pump that continuously pumpsthe GDNF fluid into the brain. However, this highly invasive procedurewas found to lead to toxic effects in the brain, was not effective owingto lack of penetration of the GDNF to the nigra-striatal tract, and wasabandoned. These failed trans-cranial approaches to the treatment of PDwith GDNF highlighted the importance of re-engineering this neurotrophinso that the molecule can cross the BBB. The trans-BBB delivery route hastwo unique advantages. First, the GDNF can be given by a non-invasivesystemic injection, such as an intravenous, subcutaneous, orintra-muscular injection, and no neurosurgical procedure is required.Second, the GDNF enters the brain via the trans-vascular route. Sinceevery neuron in the brain is perfused by its own blood vessel, the GDNFis delivered to every cell comprising the nigra-striatal tract of brain.The GDNF could be given on a weekly or bi-weekly basis to patients withPD at doses ranging from 1, 3, 10, 30, or 100 mg. The considerations inExample 7 suggest that 25-30 mg may be a preferred dose.

Example 18

Acute and Chronic Treatment of Stroke with the HIRMAb-GDNF FusionProtein

GDNF is highly neuroprotective in experimental stroke (Kitagawa et al,Stroke 29:1417-1422 (1998)). Activation of the GDNF receptor, GFRα1,inhibits apoptosis in the neuron that is induced by an ischemic event.The demonstration of GDNF's neuroprotective effects in experimentalstroke have uniformly involved intra-cerebral injection of theneurotrophin, because the GDNF does not cross the BBB, and because theBBB is intact in the first 5 hours after stroke, when the rescue ofdying neurons is still possible. The HIRMAb-GDNF is a re-engineered formof GDNF that is both equi-potent with GDNF as a neuroprotective agent,and is able to cross the human BBB following systemic administration.Thus, the patient suffering from an acute stroke could be rapidlytreated with intravenous HIRMAb-GDNF in the emergency room. TheHIRMAb-GDNF fusion protein will rapidly enter the brain and activateGFRα1 receptors on neurons to inhibit the apoptosis cycle induced by thestroke. In addition, chronic treatment with systemic HIRMAb-GDNF fusionprotein could be therapeutic in the rehabilitation phase of stroke,since the intra-cerebral infusion of GDNF into the post-stroke braininduces neurogenesis, and neural repair in the post-stroke period(Kobayashi et al, 2006).

Example 19

Treatment of Motor Neuron Disease with the HIRMAb-GDNF Fusion Protein

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) andspinal muscular atrophy (SMA) cause progressive paralysis leading topremature death. GDNF is highly neuroprotective of spinal cord motorneurons (Bohn supra). However, it is unlikely that GDNF will bedeveloped as a drug for ALS or SMA, given the failed clinical trials ofother neurotrophic factors for these conditions. Both BDNF and ciliaryneurotrophic factor (CNTF) were administered to ALS patients in the1990s by subcutaneous administration. The subcutaneous administrationphase III clinical trials failed, because BDNF and CNTF, like GDNF, donot cross the BBB. However, with the re-engineering of GDNF as theHIRMAb-GDNF fusion protein demonstrated herein, conditions such as ALSor SMA could now be amenable to treatment by systemic administration.The HIRMAb fusion proteins cross the blood-spinal cord barrier, which asbeen demonstrated by brain scanning of adult Rhesus monkeys followingintravenous administration of the HIRMAb fusion protein (Boado et al,Biotechnol. Bioeng, 97(6):1376-1386 (2007); Boado et al, Bioconjug.Chem. 18:447-455 (2007)).

Example 20

Treatment of Brain or Spinal Cord Injury with the HIRMAb-GDNF FusionProtein

GDNF promotes neural repair in the period following acute experimentalbrain injury, such as with a lateral fluid percussion injury (Bakshi etal, Eur. J. Neurosci. 23:2119-2134 (2006)). GDNF does not cross the BBB,and the BBB is intact in the days/weeks after head injury, when thebrain attempts to heal from the acute insult. Therefore, the beneficialeffects of GDNF on acute head injury could only be demonstrated by theintra-cerebral injection of genetically modified stem cells that secreteGDNF. Although the trans-cranial injection approach may be feasible inthe rat brain, the human brain is 1000-fold larger than a rat brain.Diffusion of the GDNF from the depot injection site becomes limiting,and the GDNF cannot penetrate to the wound area, beyond the localinjection site. Alternatively, if the GDNF is delivered to brain via thetrans-vascular route, then every injured neuron in the brain is exposedto the GDNF. The latter is feasible with the weekly, bi-weekly, or dailysystemic administration of the HIRMAb-GDNF fusion protein during thepost-injury repair and rehabilitation period.

Similarly, motor neurons of the spinal cord are responsive to GDNF, andGDNF can promote neural repair following experimental spinal cord injury(Lu et al, J. Neurotrauma 19:1081-1090 (2002)). However, because theopening of the BBB following spinal cord injury is only transient, it isnot possible to treat spinal cord injury with systemic GDNFadministration. Instead, the GDNF expressing plasmid DNA was injecteddirectly into the rat spinal cord following the acute spinal cordinjury. However, the size of the spinal cord in humans is 1000-foldlarger than that of the rat. Therefore, it is not possible to distributeGDNF to the entire lesioned area following direct spinal injection ofthe GDNF therapeutic in humans Alternatively, GDNF is delivered to allinjured neurons in the spinal cord following a trans-vascular delivery,and this is possible with the systemic administration of the HIRMAb-GDNFfusion protein.

Example 21

Treatment of Drug and Alchohol Addiction with the HIRMAb-GDNF FusionProtein

The intra-cerebral injection of GDNF results in treatment of animalsaddicted to either opioid, or non-opioid drugs. With respect tonon-opioid drugs, the intra-cerebral injection of GDNF decreases cocaineself-administration in rats (Green-Sadan et al, Eur. J. Neurosci., 18:2093-2098, 2003). Similarly, the intra-cerebral injection of GDNFdecreases ethanol self-administration (He et al, J. Neurosci., 25:619-628, 2005). The GDNF has to be administered via a trans-cranialroute, because GDNF does not cross the BBB. The drug, ibogaine,decreases withdrawal symptoms to chronic opioid drugs, cocaine, oralchohol, and works by increasing GDNF in regions of the brain such asthe ventral tegmental area (VTA) (He and Ron, FASEB J, 20: E1820-D1827,2006). Although ibogaine is a small molecule that crosses the BBB, itsapplication in the treatment of addiction is limited by severe sideeffects. GDNF is implicated in another study related to cocaineaddiction. Cocaine increases tyrosine hydroxylase in the VTA, and thiseffect of cocaine is blocked by the intra-cerebral injection of GDNF(Messer et al, Neuron, 26: 247-257, 2000). With respect tomethamphetamine addiction, a decrease in brain concentration of GDNF,such as occurs in GDNF knock-out heterozygote mice, results in increasedmethamphetamine self-administration (Yan et al, FASEB J, 21: 1994-2004,2007). These forms of addiction could be treated by systemicadministration of GDNF if this neurotrophin was re-formulated to crossthe BBB, which is the case for the HIRMAb-GDNF fusion protein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

SEQUENCES <210>  1 <211> 36 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Oligonucleotide<400> 1 cctgtctccg ggtaaatatt tgcgacggcc ggcaag <210>  2 <211> 36 <212>DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 2cttgccggcc gtcgcaaata tttacccgga gacagg <210>  3 <211> 42 <212> DNA<213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 3atgctcgagg aattcccatg gatgatggct agcaagctta tg <210>  4 <211> 42 <212>DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 4cataagcttg ctagccatca tccatgggaa ttcctcgagc at <210>  5 <211> 56 <212>DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Oligonucleotide <400> 5tccggatcct cgcgagtatg cactctgacc ctgcccgtcg aggtgagctg agcgtg <210>  6<211> 56 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Oligonucleotide <400> 6cacgctcagc tcacctcgac gggcagggtc agagtgcata ctcgcgagga tccgga <210>  7<211> 119 <212> DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 7agtcgtacgt gcgggccctt accatggata gcaaaaagag aattggctgg cgattcataaggatagacac ttcttgtgta tgtacattga ccattaaaag gtgatcgcga ctcgagatg <210> 8 <211> 119 <212> DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 8catctcgagt cgcgatcacc ttttaatggt caatgtacat acacaagaag tgtctatccttatgaatcgc cagccaattc tctttttgct atccatggta agggcccgca cgtacgact <210>  9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223>SDescription of Artificial equence: Synthetic Oligonucleotide <400> 9atctcgcgag tatgcactct gaccctgcc <210> 10 <211> 27 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 10atctcgcgat caccttttaa tggtcaa <210> 11 <211> 42 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 11atggctagcg atatcggtac cgtatacgga tccctcgaga tg <210> 12 <211> 42 <212>DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 12catctcgagg gatccgtata cggtaccgat atcgctagcc at <210> 13 <211> 25 <212>DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400>  13gtgacaaaca cagacatagg atatc <210> 14 <211> 24 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 14atgctcgagc taacactctc ccct <210> 15 <211> 26 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 15atgaatattc caccatggaa tgcagc <210> 16 <211> 27 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 16ataggatcct caccttttaa tggtcaa <210> 17 <211> 42 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 17aaaaggccag gaaccgaatt cagatctcgt tgctggcgtt tt <210> 18 <211> 42 <212>DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 18aaaacgccag caacgagatc tgaattcggt tcctggcctt tt <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 19atcgaattca agcttgcggc cgcgtataca gatctatc <210> 20 <211> 38 <212> DNA<213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 20gatagatctg tatacgcggc cgcaagcttg aattcgat <210> 21 <211> 378 <212> DNA<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic <220> <221> CDS <222>(1)..(375) <400> 21ctg tct ccg ggt aaa tcg agt atg cac tct gac cct gcc cgt cga ggtLeu Ser Pro Gly Lys Ser Ser Met His Ser Asp Pro Ala Arg Arg Glygag ctg agc gtg tgt gac agt att agt gag tgg gta acg gcg gca gacGlu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala Aspaaa aag act gca gtg gac atg tcg ggc ggg acg gtc aca gtc ctt gaaLys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu Gluaag gtc cct gta tca aaa ggc caa ctg aag caa tac ttc tac gag accLys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu Thraag tgc aat ccc atg ggt tac aca aaa gaa ggc tgc agg ggc ata gacLys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Aspaaa agg cat tgg aac tcc cag tgc cga act acc cag tcg tac gtg cggLys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arggcc ctt acc atg gat agc aaa aag aga att ggc tgg cga ttc ata aggAla Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile Argata gac act tct tgt gta tgt aca ttg acc att aaa agg tgaIle Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg <210> 22 <211> 125<212> PRT <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 22Leu Ser Pro Gly Lys Ser Ser Met His Ser Asp Pro Ala Arg Arg GlyGlu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala AspLys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu GluLys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu ThrLys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile AspLys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val ArgAla Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile ArgIle Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg <210> 23 <211> 2711<212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 23tagtctttct cttcagtgac aaacacagac ataggatatt ccaccatgga atgcagctgggtcatgctct tcctcctgtc aggaactgca ggtgtccatt gccaggttca gctgcagcagtctggacctg agctggtgaa gcctggggct ttagtgaaga tatcctgcaa ggcttctggttacaccttca caaactacga tatacactgg gtgaagcaga ggcctggaca gggacttgagtggattggat ggatttatcc tggagatggt agtactaagt acaatgagaa attcaagggcaaggccacac tgactgcaga caaatcctcc agcacagcct acatgcacct cagcagcctgacttctgaga aatctgcagt ctatttctgt gcaagagagt gggcttactg gggccaagggactctggtca ctgtctctgc agctagcacc aagggcccat cggtcttccc cctggcaccctcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttccccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttcccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctccagcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaaggtggacaaga aagttggtga gaggccagca cagggaggga gggtgtctgc tggaagccaggctcagcgct cctgcctgga cgcatcccgg ctatgcagcc ccagtccagg gcagcaaggcaggccccgtc tgcctcttca cccggaggcc tctgcccgcc ccactcatgc tcagggagagggtcttctgg ctttttcccc aggctctggg caggcacagg ctaggtgccc ctaacccaggccctgcacac aaaggggcag gtgctgggct cagacctgcc aagagccata tccgggaggaccctgcccct gacctaagcc caccccaaag gccaaactct ccactccctc agctcggacaccttctctcc tcccagattc cagtaactcc caatcttctc tctgcagagc ccaaatcttgtgacaaaact cacacatgcc caccgtgccc aggtaagcca gcccaggcct cgccctccagctcaaggcgg gacaggtgcc ctagagtagc ctgcatccag ggacaggccc cagccgggtgctgacacgtc cacctccatc tcttcctcag cacctgaact cctgggggga ccgtcagtcttcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacatgcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacggcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtaccgtgtggtcag gtgtggtcag ggtcctcacc aggactggct gaatggcaag gagtacaagtgcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaaggtgggacccg tggggtgcga gggccacatg gacagaggcc ggctcggccc accctctgccctgagagtga ccgctgtacc aacctctgtc cctacagggc agccccgaga accacaggtgtacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctggtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggagaacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagcaagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatgcatgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatcgagtatgcact ctgaccctgc ccgtcgaggt gagctgagcg tgtgtgacag tattagtgagtgggtaacgg cggcagacaa aaagactgca gtggacatgt cgggcgggac ggtcacagtccttgaaaagg tccctgtatc aaaaggccaa ctgaagcaat acttctacga gaccaagtgcaatcccatgg gttacacaaa agaaggctgc aggggcatag acaaaaggca ttggaactcccagtgccgaa ctacccagtc gtacgtgcgg gcccttacca tggatagcaa aaagagaattggctggcgat tcataaggat agacacttct tgtgtatgta cattgaccat taaaaggtgatcgattttgc gacggccggc aagcccccgc tccccgggct ctcgcggtcg cacgaggatgcttggcacgt accccctgta catacttccc gggcgcccag catggaaata aagcacccagcgctgccctg ggcccctgcg agactgtgat ggttctttcc acgggtcagg ccgagtctgaggcctgagtg gcatgaggga ggcagagcgg gtcccactgt ccccacactg gcccaggctgtgcaggtgtg cctgggccgc ctagggtggg gctcagccag gggctgccct cggcagggtgggggatttgc c <210> 24 <211> 582 <212> PRT <213> Artificial Sequence<220> <223>  Description of Artificial Sequence: Synthetic Protein <400>24 Met Glu Cys Ser trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala GlyVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr PheThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly LeuGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser SerThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala ValTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Arg ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser SerMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp SerIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp MetSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys GlyGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly TyrThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser GlnCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser LysLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val CysThr Leu Thr Ile Lys Arg <210> 25 <211> 582 <212> PRT <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 25Met Glu Cys Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala GlyVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr PheThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly LeuGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser SerThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala ValTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser SerMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp SerIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp MetSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys GlyGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly TyrThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser GlnCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser LysLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val CysThr Leu Thr He Lys Arg <210> 26 <211> 1757 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 26attccaccat ggaatgcagc tgggtcatgc tcttcctcct gtcaggaact gcaggtgtccattgccaggt tcagctgcag cagtctggac ctgagctggt gaagcctggg gctttagtgaagatatcctg caaggcttct ggttacacct tcacaaacta cgatatacac tgggtgaagcagaggcctgg acagggactt gagtggattg gatggattta tcctggagat ggtagtactaagtacaatga gaaattcaag ggcaaggcca cactgactgc agacaaatcc tccagcacagcctacatgca cctcagcagc ctgacttctg agaaatctgc agtctatttc tgtgcaagagagtgggctta ctggggccaa gggactctgg tcactgtctc tgcagctagc accaagggcccatcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgggctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccctgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctcagcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtgaatcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaactcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctcttccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtggtggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtggtcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaaggtctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagccccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccaggtcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggagagcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggctccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtcttctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccctgtctccggg taaatcgagt atgcactctg accctgcccg tcgaggtgag ctgagcgtgtgtgacagtat tagtgagtgg gtaacggcgg cagacaaaaa gactgcagtg gacatgtcgggcgggacggt cacagtcctt gaaaaggtcc ctgtatcaaa aggccaactg aagcaatacttctacgagac caagtgcaat cccatgggtt acacaaaaga aggctgcagg ggcatagacaaaaggcattg gaactcccag tgccgaacta cccagtcgta cgtgcgggcc cttaccatggatagcaaaaa gagaattggc tggcgattca taaggataga cacttcttgt gtatgtacattgaccattaa aaggtga <210> 27 <211> 1749 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <220> <221> CDS <222>(1)..(1746) <400> 27atg gaa tgc agc tgg gtc atg ctc ttc ctc ctg tca gga act gca ggtMet Glu Cys Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala Glygtc cat tgc cag gtt cag ctg cag cag tct gga cct gag ctg gtg aagVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lyscct ggg gct tta gtg aag ata tcc tgc aag gct tct ggt tac acc ttcPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Pheaca aac tac gat ata cac tgg gtg aag cag agg cct gga cag gga cttThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leugag tgg att gga tgg att tat cct gga gat ggt agt act aag tac aatGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asngag aaa ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agcGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Seraca gcc tac atg cac ctc agc agc ctg act tct gag aaa tct gca gtcThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Valtat ttc tgt gca aga gag tgg gct tac tgg ggc caa ggg act ctg gtcTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu Valact gtc tct gca gct agc acc aag ggc cca tcg gtc ttc ccc ctg gcaThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Alaccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctgPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leugtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggcVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Glygcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tcaAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Sergga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttgGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leuggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac accGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thraag gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac acaLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thrtgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttcCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phectc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cctLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Progag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtcGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Valaag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag acaLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thraag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtcLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val ser Valctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgcLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cysaag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tccLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Seraaa gcc aaa ggg cag CCC cga gaa cca cag gtg tac acc ctg ccc ccaLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Protcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtcSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr cys Leu Valaaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat gggLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Glycag ccg gag aac aac tac aag acc acg cct CCC gtg ctg gac tcc gacGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Aspggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tggGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trpcag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cacGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu Hisaac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tcg agtAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser pro Gly Lys Ser Seratg cac tct gac cct gcc cgt cga ggt gag ctg agc gtg tgt gac agtMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Seratt agt gag tgg gta acg gcg gca gac aaa aag act gca gtg gac atgIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Mettcg ggc ggg acg gtc aca gtc ctt gaa aag gtc cct gta tca aaa ggcSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Glycaa ctg aag caa tac ttc tac gag acc aag tgc aat ccc atg ggt tacGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyraca aaa gaa ggc tgc agg ggc ata gac aaa agg cat tgg aac tcc cagThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Glntgc cga act acc cag tcg tac gtg cgg gcc ctt acc atg gat agc aaaCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lysaag aga att ggc tgg cga ttc ata agg ata gac act tct tgt gta tgtLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cysaca ttg acc att aaa agg tga Thr Leu Thr Ile Lys Arg <210> 28 <211> 582<212> PRT <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 28Met Glu Cys Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala GlyVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr PheThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro gly Gln Gly LeuGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser SerThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala ValTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp gly Gln Gly Thr Leu ValThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His glu Asp Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser SerMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp SerIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp MetSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys GlyGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly TyrThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser GlnCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser LysLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val CysThr Leu Thr Ile Lys Arg <210> 29 <211> 714 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 29gatatcacca tggagacaga cacactcctg ctatggctct tgttgctcat gtttccaggtaccagatgtg acatccagat gacccagtct ccatcctcct tatctgcctc tctgggagaaagagtcagtc tcacttgtcg ggcaagtcag gacattggtg gtaacttata ctggcttcagcagggaccag atggaactat taaacgcctg atctacgcca catccagttt agattctggtgtccccaaaa ggttcagtgg cagtaggtct gggtcagatt attctctcac catcagcagccttgagtctg aagattttgt agactattac tgtctacagt attctagttc tccgtggacgttcggtggag cgacaaagat ggaaataaaa cgaactgtgg ctgcaccatc tgtcttcatcttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaataacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggtaactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagcaccctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacccatcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttag <210> 30<211> 705 <212> DNA <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <220> <221>  CDS <222>(1).. (702) <400>  30atg gag aca gac aca ctc ctg cta tgg ctc ttg ttg ctc atg ttt ccaMet Glu Thr Asp Thr Leu Leu Leu Trp Leu Leu Leu Leu Met Phe Proggt acc aga tgt gac atc cag atg acc cag tct cca tcc tcc tta tctGly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Sergcc tct ctg gga gaa aga gtc agt ctc act tgt cgg gca agt cag gacAla Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Aspatt ggt ggt aac tta tac tgg ctt cag cag gga cca gat gga act attIle Gly Gly Asn Leu Tyr Trp Leu Gln Gln Gly Pro Asp Gly Thr Ileaaa cgc ctg atc tac gcc aca tcc agt tta gat tct ggt gtc ccc aaaLys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lysagg ttc agt ggc agt agg tct ggg tca gat tat tct ctc acc atc agcArg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Seragc ctt gag tct gaa gat ttt gta gac tat tac tgt cta cag tat tctSer Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Seragt tct ccg tgg acg ttc ggt gga gcg aca aag atg gaa ata aaa cgaSer Ser Pro Trp Thr Phe Gly Gly Ala Thr Lys Met Glu Ile Lys Argact gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cagThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Glnttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tatLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyrccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcgPro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Serggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc accGly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thrtac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaaTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lyscac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cccHis Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Progtc aca aag agc ttc aac agg gga gag tgt tagVal Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 31 <211> 234 <212> PRT<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Protein <400> 31Met Glu Thr Asp Thr Leu Leu Leu Trp Leu Leu Leu Leu Met PheGly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser LeuAla Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser GlnIle Gly Gly Asn Leu Tyr Trp Leu Gln Gln Gly Pro Asp Gly ThrLys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val ProArg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr IleSer Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln TyrSer Ser Pro Trp Thr Phe Gly Gly Ala Thr Lys Met Glu Ile LysThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp GluLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn PhePro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnGly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr GluHis Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser SerVal Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 32 <211> 6505 <212> DNA<213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 32cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatcaattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggtaaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgtatgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacggtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattgacgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactttcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttggcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccccattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcgtaacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatataagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatacgactcactat agggagaccc ttgctagcga tattccacca tggaatgcag ctgggtcatgctcttcctcc tgtcaggaac tgcaggtgtc cattgccagg ttcagctgca gcagtctggacctgagctgg tgaagcctgg ggctttagtg aagatatcct gcaaggcttc tggttacaccttcacaaact acgatataca ctgggtgaag cagaggcctg gacagggact tgagtggattggatggattt atcctggaga tggtagtact aagtacaatg agaaattcaa gggcaaggccacactgactg cagacaaatc ctccagcaca gcctacatgc acctcagcag cctgacttctgagaaatctg cagtctattt ctgtgcaaga gagtgggctt actggggcca agggactctggtcactgtct ctgcagctag caccaagggc ccatcggtct tccccctggc accctcctccaagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaaccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggctgtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagcttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggacaagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacctgaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatgatctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgaggtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgggaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggactggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatcgagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgcccccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttctatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaagaccacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtggacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctgcacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatcgag tatgcactctgaccctgccc gtcgaggtga gctgagcgtg tgtgacagta ttagtgagtg ggtaacggcggcagacaaaa agactgcagt ggacatgtcg ggcgggacgg tcacagtcct tgaaaaggtccctgtatcaa aaggccaact gaagcaatac ttctacgaga ccaagtgcaa tcccatgggttacacaaaag aaggctgcag gggcatagac aaaaggcatt ggaactccca gtgccgaactacccagtcgt acgtgcgggc ccttaccatg gatagcaaaa agagaattgg ctggcgattcataaggatag acacttcttg tgtatgtaca ttgaccatta aaaggtgagg atccctcgagcatgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagcctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttccttgaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgcattgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaagggggaggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggcggaaagaacc agtggcggta atacggttat ccacagaatc aggggataac gcaggaaagaacatgtgagc aaaaggccag caaaaggcca ggaaccgaat tcgatattcc atacacatacttctgtgttc ctttgaaagc tggacttttg caggctccac cagacctctc tagatcaattcctttgccta atttcgctta caatttacgc gcgcgttgac attgattatt gactagttattaatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttacataacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtcaataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtggagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacgccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgaccttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtgatgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttccaagtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggactttccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgggaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatcgaaattaata cgactcacta tagggagacc caagctggct agcgatatca ccatggagacagacacactc ctgctatggc tcttgttgct catgtttcca ggtaccagat gtgacatccagatgacccag tctccatcct ccttatctgc ctctctggga gaaagagtca gtctcacttgtcgggcaagt caggacattg gtggtaactt atactggctt cagcagggac cagatggaactattaaacgc ctgatctacg ccacatccag tttagattct ggtgtcccca aaaggttcagtggcagtagg tctgggtcag attattctct caccatcagc agccttgagt ctgaagattttgtagactat tactgtctac agtattctag ttctccgtgg acgttcggtg gagcgacaaagatggaaata aaacgaactg tggctgcacc atctgtcttc atcttcccgc catctgatgagcagttgaaa tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagagaggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgtcacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaaagcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctcgcccgtcaca aagagcttca acaggggaga gtgttagctc gagtctagag ggcccgtttaaacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctcccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatgaggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggcaggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctctatggcttct gaggcggaaa gaaccagtgg cggtaatacg gttatccaca gaatcaggggataacgaaat gaggacttaa cctgtggaaa tatcaagctt gcggccgcgt atcgacgctctcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcaccgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggggcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatcttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgatgccggccacg atgcgtccgg cgtagaggat ctctgacgga aggaaagaag tcagaaggcaaaaacgagag taactccaca gtagctccaa attctttata agggtcaatg tccatgccccaaagccaccc aaggcacagc ttggaggctt gaacagtggg acatgtacaa gagatgattaggcagaggtg aaaaagttgc atggtgctgg tgcgcagacc aatttatgcc tacagcctcctaatacaaag acctttaacc taatctcctc ccccagctcc tcccagtcct taaacacacagtctttgaag taggcctcaa ggtcggtcgt tgacattgct gggagtccaa gagtcctcttatgtaagacc ttgggcagga tctgatgggc gttcacggtg gtctccatgc aacgtgcagaggtgaagcga agtgcacacg gaccggcaga tgagaaggca cagacgggga gaccgcgtaaagagaggtgc gccccgtggt cggctggaac ggcagacgga gaaggggacg agagagtcccaagcggcccc gcgaggggtc gtccgcggga ttcagcgccg acgggacgta aacaaaggacgtcccgcgaa ggatctaaag ccagcaaaag tcccatggtc ttataaaaat gcatagctttaggaggggag cagagaactt gaaagcatct tcctgttagt ctttcttctc gtagacttcaaacttatact tgatgccttt ttcctcctgg acctcagaga ggacgcctgg gtattctgggagaagtttat atttccccaa atcaatttct gggaaaaacg tgtcactttc aaattcctgcatgatccttg tcacaaagag tctgaggtgg cctggttgat tcatggcttc ctggtaaacagaactgcctc cgactatcca aaccatgtct actttacttg ccaattccgg ttgttcaataagtcttaagg catcatccaa acttttggca agaaaatgag ctcctcgtgg tggttctttgagttctctac tgagaactat attaattctg tcctttaaag gtcgattctt ctcaggaatggagaaccagg ttttcctacc cataatcacc agattctgtt taccttccac tgaagaggttgtggtcattc tttggaagta cttgaactcg ttcctgagcg gaggccaggg tcggtctccgttcttgccaa tccccatatt ttgggacacg gcgacgatgc agttcaatgg tcgaaccatgatggcaaatt ctagaatcga taagcttttt gcaaaagcct aggcctccaa aaaagcctcctcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaaaattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatgggcggagttagg ggcgggacta tggttgctga ctaattgaga tgcagatctc gagctagcacgcgtaagagc tcggtacctc cctac  <210> 33 <211> 1749 <212> DNA <213>Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <220> <221> CDS <222> (1)..(1746) <400> 33atg gaa tgc agc tgg gtc atg ctc ttc ctc ctg tca gga act gca ggtMet Glu Cys Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala Glygtc cat tgc cag gtt cag ctg cag cag tct gga cct gag ctg gtg aagVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lyscct ggg gct tta gtg aag ata tcc tgc aag gct tct ggt tac acc ttcPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Pheaca aac tac gat ata cac tgg gtg aag cag agg cct gga cag gga cttThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leugag tgg att gga tgg att tat cct gga gat ggt agt act aag tac aatGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asngag aaa ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agcGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Seraca gcc tac atg cac ctc agc agc ctg act tct gag aaa tct gca gtcThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Valtat ttc tgt gca aga gag tgg gct tac tgg ggc caa ggg act ctg gtcTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu Valact gtc tct gca gct agc acc aag ggc cca tcg gtc ttc ccc ctg gcaThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Alaccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctgPro ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leugtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggcVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Glygcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tcaAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Sergga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttgGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leuggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac accGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thraag gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac acaLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thrtgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttcCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phectc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cctLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Progag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtcGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Valaag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag acaLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thraag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtcLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Valctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgcLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cysaag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tccLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Seraaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc ccaLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Protcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtcSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Valaaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat gggLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Glycag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gacGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Aspggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tggGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trpcag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cacGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu Hisaac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tcg agtAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Seratg cac tct gac cct gcc cgt cga ggt gag ctg agc gtg tgt gac agtMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Seratt agt gag tgg gta acg gcg gca gac aaa aag act gca gtg gac atgIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Mettcg ggc ggg acg gtc aca gtc ctt gaa aag gtc cct gta tca aaa ggcSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Glycaa ctg aag caa tac ttc tac gag acc aag tgc aat ccc atg ggt tacGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyraca aaa gaa ggc tgc agg ggc ata gac aaa agg cat tgg aac tcc cagThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Glntgc cga act acc cag tcg tac gtg cgg gcc ctt acc atg gat agc aaaCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lysaag aga att ggc tgg cga ttc ata agg ata gac act tct tgt gta tgtLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cysaca ttg acc att aaa agg tga Thr Leu Thr Ile Lys Arg <210> 34 <211> 582<212> PRT <213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 34Met Glu Cys Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr Ala GlyVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysPro Gly Ala Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr PheThr Asn Tyr Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly LeuGlu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser SerThr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala ValTyr Phe Cys Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser SerMet His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp SerIle Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp MetSer Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys GlyGln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly TyrThr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser GlnCys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser LysLys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val CysThr Leu Thr Ile Lys Arg         Thr Leu Thr He Lys Arg <210> 35 <211>705 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221>  CDS <222>(1).. (702) <400> 35atg gag aca gac aca ctc ctg cta tgg ctc ttg ttg ctc atg ttt ccaMet Glu Thr Asp Thr Leu Leu Leu Trp Leu Leu Leu Leu Met Phe Proggt acc aga tgt gac atc cag atg acc cag tct cca tcc tcc tta tctGly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Sergcc tct ctg gga gaa aga gtc agt ctc act tgt cgg gca agt cag gacAla Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp att ggt ggt aac tta tac tgg ctt cag cag gga cca gat gga act attIle Gly Gly Asn Leu Tyr Trp Leu Gln Gln Gly Pro Asp Gly Thr Ileaaa cgc ctg atc tac gcc aca tcc agt tta gat tct ggt gtc ccc aaaLys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lysagg ttc agt ggc agt agg tct ggg tca gat tat tct ctc acc atc agcArg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Seragc ctt gag tct gaa gat ttt gta gac tat tac tgt cta cag tat tctSer Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Seragt tct ccg tgg acg ttc ggt gga gcg aca aag atg gaa ata aaa cgaSer Ser Pro Trp Thr Phe Gly Gly Ala Thr Lys Met Glu Ile Lys Argact gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cagThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Glnttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tatLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyrccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcgPro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Serggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc accGly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thrtac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaaTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lyscac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cccHis Lys Val Tyr ala cys Glu Val Thr His Gln gly Leu Ser Ser Progtc aca aag ac ttc aac agg gga gag tgt tagVal Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 36 <211> 234 <212> PRT<213> Artificial Sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 36Met Glu Thr Asp Thr Leu Leu Leu Trp Leu Leu Leu Leu Met Phe ProGly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln AspIle Gly Gly Asn Leu Tyr Trp Leu Gln Gln Gly Pro Asp Gly Thr IleLys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro LysArg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile SerSer Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr SerSer Ser Pro Trp Thr Phe Gly Gly Ala Thr Lys Met Glu Ile Lys ArgThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu GlnLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln SerGly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser ProVal Thr Lys Ser Phe Asn Arg Gly Glu Cys       <210> 37 <211> 564 <212>DNA <213> Mus musculus <220> <221> CDS <222> (1)..(561) <400> 37atg gtt cga cca ttg aac tgc atc gtc gcc gtg tcc caa aat atg gggMet Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Glyatt ggc aag aac gga gac cga ccc tgg cct ccg ctc agg aac gag ttcIle Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg Asn Glu Pheaag tac ttc caa aga atg acc aca acc tct tca gtg gaa ggt aaa cagLys Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Glnaat ctg gtg att atg ggt agg aaa acc tgg ttc tcc att cct gag aagAsn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lysaat cga cct tta aag gac aga att aat ata gtt ctc agt aga gaa ctcAsn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leuaaa gaa cca cca cga gga gct cat ttt ctt gcc aaa agt ttg gat gatLys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Aspgcc tta aga ctt att gaa caa ccg gaa ttg gca agt aaa gta gac atgAla Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp Metgtt tgg ata gtc gga ggc agt tct gtt tac cag gaa gcc atg aat caaVal Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Glncca ggc cac ctc aga ctc ttt gtg aca agg atc atg cag gaa ttt gaaPro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Gluagt gac acg ttt ttc cca gaa att gat ttg ggg aaa tat aaa ctt ctcSer Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leucca gaa tac cca ggc gtc ctc tct gag gtc cag gag gaa aaa ggc atcPro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ileaag tat aag ttt gaa gtc tac gag aag aaa gac taaLys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp <210> 38 <211> 187 <212> PRT<213> Mus musculus <400> 38Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met GlyIle Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg Asn Glu PheLys Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys GlnAsn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu LysAsn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu LeuLys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp AspAla Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp MetVal Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met Asn GlnPro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe GluSer Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu LeuPro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly IleLys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp <210> 39 <211> 119 <212> PRT<213> Homo sapiens <400> 39His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser IleSer Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met SerGly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Gly GlnLeu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr ThrLys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln CysArg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys LysArg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys ThrLeu Thr Ile Lys Arg Gly Arg <210> 40 <211> 25 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 40ATGAAGTTATGGGATGTCGTGGCTG <210> 41 <211> 25 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 41TCAGATACATCCACACCTTTTAGCG <210> 42 <211> 26 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Oligonucleotide <400> 42CATCACCAGATAAACAAATGGCAGTG <210> 43 <211> 636 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 43ATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGGTAAGAGGCCTCCCGAGGCGCCCGCCGAAGACCGCTCCCTCGGCCGCCGCCGCGCGCCCTTCGCGCTGAGCAGTGACTCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGA<210> 44 <211> 211 <212> PRT <213> artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 44MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI <210> 45 <211> 1797<212> DNA <213> artificial sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 45ATGGACTGGACCTGGAGGGTGTTCTGCCTGCTTGCAGTGGCCCCCGGAGCCCACAGCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAACTACGATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACTTCTGAGAAATCTGCAGTCTATTTCTGTGCAAGAGAGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAGTTCATCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGA <210> 46 <211> 598 <212> PRT <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 46MDWTWRVFCLLAVAPGAHSQVQLQQSGPELVKPGALVKISCKASGYTFTNYDIHWVKQRPGQGLEWIGWIYPGDGSTKYNEKFKGKATLTADKSSSTAYMHLSSLTSEKSAVYFCAREWAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSSSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI <210> 47 <211> 6342 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 47GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGGCCGCCACCATGGAGACCCCCGCCCAGCTGCTGTTCCTGTTGCTGCTTTGGCTTCCAGATACTACCGGCGACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTGGTAACTTATACTGGCTTCAGCAGGGACCAGATGGAACTATTAAACGCCTGATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACTATTACTGTCTACAGTATTCTAGTTCTCCGTGGACGTTCGGTGGAGGCACAAAGATGGAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGAGCGGCCGCCACTGTGCTGGAGCCGCCACCATGGACTGGACCTGGAGGGTGTTCTGCCTGCTTGCAGTGGCCCCCGGAGCCCACAGCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAACTACGATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACTTCTGAGAAATCTGCAGTCTATTTCTGTGCAAGAGAGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAGTTCATCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGAAACCCGAGCTCGGTACCAAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGGGGAGGTACCGAGCTCTTACGCGTGCTAGCTCGAGATCTGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTATCGATTCTAGAAGCCGCCACCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTCAGACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCCTTCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCGCGGGGCCGCTTGGGACTCTCTCGTCCCCTTCTCCGTCTGCCGTTCCAGCCGACCACGGGGCGCACCTCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGTCCGTGTGCACTTCGCTTCACCTCTGCACGTTGCATGGAGACCACCGTGAACGCCCATCAGATCCTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCCCAGCAATGTCAACGACCGACCTTGAGGCCTACTTCAAAGACTGTGTGTTTAAGGACTGGGAGGAGCTGGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCG <210>48 <211> 234 <212> PRT <213> artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 48METPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASLGERVSLTCRASQDIGGNLYWLQQGPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKMEIKRTVAAPSVFIFPPSDEQLKSGTASvvCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC <210> 49 <211> 598 <212> PRT <213> artificial sequence<220> <223> Description of Artificial Sequence: Synthetic Protein <400>49MDWTWRVFCLLAVAPGAHSQVQLQQSGPELVKPGALVKISCKASGYTFTNYDIHWVKQRPGQGLEWIGWIYPGDGSTKYNEKFKGKATLTADKSSSTAYMHLSSLTSEKSAVYFCAREWAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSSSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI <210> 50 <211> 187 <212> PRT <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 50MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD <210> 51 <211> 405 <212> DNA <213>artificial sequence <220> <223>Description of Artificial Sequence: Synthetic DNA <400> 51TCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGA<211> 134 <212> PRT <213> artificial sequence <220> <223>Description of Artificial Sequence: Synthetic Protein <400> 52SPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGOACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI

1.-28. (canceled)
 29. A composition comprising alpha-L-iduronidase(IDUA) covalently linked to the amino terminus of an antigen bindingfragment of an antibody or to an antibody fragment binding to atransferrin receptor that is capable of crossing the blood brain barrier(BBB), wherein the composition is capable of producing an averageelevation of concentration in the brain of the IDUA of at least about 1ng/gram brain following intravenous administration, and wherein thecomposition comprises a mineral acid salt or a salt of an organic acid.